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2024 – N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO
Patchliner Publication in The Journal of Membrane Biology (2024) Authors: Brockmöller S., Molitor L.M., Seeger T., Worek F., Rothmiller S.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer’s or Parkinson’s, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

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2024 – Single-cell ionic current phenotyping elucidates non-canonical features and predictive potential of cardiomyocytes during automated drug experiments
Patchliner Publication in J Physiol. (2024) Authors: Clark A.P., Wei S., Christini D.J., Krogh-Madsen T.

All new drugs must go through preclinical screening tests to determine their proarrhythmic potential. While these assays effectively filter out dangerous drugs, they are too
conservative, often misclassifying safe compounds as proarrhythmic. In this study, we attempt to address this shortcoming with a novel, medium-throughput drug-screening approach: we use an automated patch-clamp system to acquire optimized voltage clamp (VC) and action potential (AP) data from human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) at several drug concentrations (baseline, 3×, 10× and 20× the effective free plasma concentrations). With our novel method, we show correlations between INa block and upstroke slowing after treatment with flecainide or quinine. Additionally, after quinine treatment, we identify significant reductions in current during voltage steps designed to isolate If and IKs. However, we do not detect any IKr block by either drug, and upon further investigation, do not see any IKr present in the iPSC-CMs when prepared for automated patch experiments (i.e. in suspension) – this is in contrast to similar experiments we have conducted with these cells using the manual patch setup. In this study, we: (1) present a proof-of-concept demonstration of a single-cell medium-throughput drug study, and (2) characterize the non-canonical electrophysiology of iPSC-CMs when prepared for experiments in a medium-throughput setting.

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2024 – Unveiling the crucial role of betaine: Modulation of GABA homeostasis via SLC6A1 transporter (GAT1)
Patchliner Pre-print Publication in bioRxiv (2024) Authors: Bhatt M., Lazzarin E., Alberto-Silva A.S., Domingo G., Zerlotti R., Gradisch R., Bazzone A., Sitte H., Stockner T., Bossi E.

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling. In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine at mM concentration acts as a slow substrate of GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. Intriguingly, when betaine is present at low concentration (0.01-3 mM) with GABA (at concentration <K0.5), it blocks the GABA reuptake. This GAT1 modulation occurs through the temporal  inhibition of the transporter, i.e., the prolonged occupancy by betaine impedes the rapid transition of the transporter to the inward conformation. The temporal inhibition results in a crucial regulatory mechanism contributing to the maintenance of GABA homeostasis, preserving neurons from excitotoxicity.

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PatchControlHT Suite – Patchliner
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2024 – Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples
Patchliner Publication in Marine Drugs (2024) Authors: Campàs M., Reverté J., Tudó À., Alkassar M., Diogène J., Sureda FX.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

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2024 – Comprehensive promotion of iPSC-CM maturation by integrating metabolic medium, nanopatterning, and electrostimulation
Patchliner Pre-print Publication in Research Square (2024) Authors: Li W., Luo X., Strano A., Arun S., Gamm O., Pötsch M., Hasse M., Steiner R., Fischer K., Pöche J., Ulbricht Y., Lesche M.

The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules, thus hindering their potential role in guiding personalised drug selection for patients. Here, we demonstrate an approach that combines lipid-enriched maturation medium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with an advanced electrophysiological, structural and metabolic phenotype. Through a systematic, stepwise parallel testing of the three stimuli, electrostimulation emerged as the pivotal factor to enhance mitochondrial development and to improve the electrophysiological properties of iPSC-CMs. The combined approach brought a substantial modification in their current composition by increasing INaItoIK1 and IKr but decreasing ICa−L, resulting in a significant change in their sensitivity to cardioactive drugs. Transcriptome analysis revealed that activation of HMCES and TFAM targets played a role in mitochondrial development, whereas the downregulation of MAPK/PI3K signalling pathways and SRF targets were associated with polyploidy of iPSC-CMs. Taken together, our study provides mechanistic insights into the maturation of iPSC-CMs with a more adult-like drug response.

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2024 – Discovery of a novel natural compound, vitekwangin B, with ANO1 protein reduction properties and anticancer potential
Patchliner Publication in Frontiers in Pharmacology (2024) Authors: Yohan S., Sion L., Minuk K., Dongguk K., Baek J.S., Raju D., Armin S., SeonJu P., Xuan N.N., Thi Thanh H.P., Oh-Bin K., Wan N., Joohan W.

Background: Prostate cancer and non-small cell lung cancer (NSCLC) present significant challenges in the development of effective therapeutic strategies. Hormone therapies for prostate cancer target androgen receptors and prostate-specific antigen markers. However, treatment options for prostatic small-cell neuroendocrine carcinoma are limited. NSCLC, on the other hand, is primarily treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors but exhibits resistance. This study explored a novel therapeutic approach by investigating the potential anticancer properties of vitekwangin B, a natural compound derived from Vitex trifolia.

Methods: Vitekwangin B was chromatographically isolated from the fruits of V. trifolia. ANO1 protein levels in prostate cancer and NSCLC cells were verified and evaluated again after vitekwangin B treatment.

Results: Vitekwangin B did not inhibit anoctamin1 (ANO1) channel function but significantly reduced ANO1 protein levels. These results demonstrate that vitekwangin B effectively inhibited cancer cell viability and induced apoptosis in prostate cancer and NSCLC cells. Moreover, it exhibited minimal toxicity to liver cells and did not affect hERG channel activity, making it a promising candidate for further development as an anticancer drug.

Conclusion: Vitekwangin B may offer a new direction for cancer therapy by targeting ANO1 protein, potentially improving treatment outcomes in patients with prostate cancer and NSCLC. Further research is needed to explore its full potential and overcome existing drug resistance challenges.

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2024 – The funnel-web spider venom derived single knot peptide Hc3a modulates acid-sensing ion channel 1a desensitisation
Patchliner Publication in Biochemical Pharmacology (2024) Authors: Budusan E., Payne C., Gonzalez T., Obergrussberger A., Becker N., Clark R., Rosengren K., Rash L., Cristofori-Armstrong B.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel involved in synaptic transmission, pain signalling, and several ischemia-associated pathological conditions. The spider venom-derived peptides PcTx1 and Hi1a are two of the most potent ASIC1a inhibitors known and have been instrumental in furthering our understanding of the structure, function, and biological roles of ASICs. To date, homologous spider peptides with different pharmacological profiles at ASIC1a have yet to be discovered. Here we report the characterisation of Hc3a, a single inhibitor cystine knot peptide from the Australian funnel-web spider Hadronyche cerberea with sequence similarity to PcTx1. We show that Hc3a has complex pharmacology and binds different ASIC1a conformational states (closed, open, and desensitised) with different affinities, with the most prominent effect on desensitisation. Hc3a slows the desensitisation kinetics of proton-activated ASIC1a currents across multiple application pHs, and when bound directly to ASIC1a in the desensitised conformation promotes current inhibition. The solution structure of Hc3a was solved, and the peptide-channel interaction examined via mutagenesis studies to highlight how small differences in sequence between Hc3a and PcTx1 can lead to peptides with distinct pharmacology. The discovery of Hc3a expands the pharmacological diversity of spider venom peptides targeting ASIC1a and adds to the toolbox of compounds to study the intricacies of ASIC1 gating.

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Mitochondrial Membrane Research
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Patchliner – Single Channel Recordings
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2024 – Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions
Patchliner and SyncroPatch 384 Publication in Frontiers in Physiology (2024) Authors: Bloothooft M., Verbruggen B., Seibertz F., van der Heyden M.A.G., Voigt N., de Boer T.P.

Background: The rapid delayed rectifier potassium current (IKr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting IKr, hERG channel, is not only permeable to K+ ions but also to Cs+ ions when present in equimolar concentrations inside and outside of the cell.

Methods: In this study, IhERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs+ or K+ as the charge carrier. Equimolar Cs+ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs+- and K+-based currents.

Results: Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs+- and K+-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs+ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs+-based conductance is larger compared to the known K+-based conductance, and the Cs+ hERG conductance can be inhibited similarly to the K+-based conductance.

Conclusion: Using equimolar Cs+ instead of K+ for IhERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small IKr current densities in combination with small membrane capacitances.

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2024 – No Influence of Asundexian on Cardiac Repolarization
Patchliner Publication in Clinical Pharmacology in Drug Development (2024) Authors: Brase C., Kanefendt F., Loewen S., Himmel H., Schmitz S.

Inhibition of activated factor XI reduces thrombogenesis while maintaining physiological hemostasis, with the expectation of reduced bleeding risk compared with standard of care in the clinical setting. Asundexian (BAY 2433334), an activated factor XI inhibitor, is in clinical development for the prevention of thromboembolic events. The effect of asundexian and its plasma metabolite M10 on cardiac repolarization and potential interactions with the hNav1.5 sodium, hCav1.2 calcium, and human ether-à-go-go-related gene (hERG) potassium channels was investigated in vitro. Additionally, asundexian effects on cardiac parameters and electrocardiogram were examined in telemetered beagle dogs. A randomized, placebo-controlled, 4-way crossover, thorough QT study in healthy adults evaluated the influence of 50 and 150 mg of asundexian on the corrected QT interval, including 400 mg of moxifloxacin as positive control. Across all studies, asundexian and M10 were not associated with any effects on cardiac repolarization. The largest in vitro effects of asundexian (approximately 20% inhibition) were seen for hCav1.2 and hERG. Throughout the thorough QT study, the upper limits of the one-sided 95% confidence interval of placebo-corrected mean changes from baseline in Fridericia corrected QT for 50 and 150 mg of asundexian were below Δ = 10 milliseconds. Asundexian demonstrated favorable safety and tolerability profiles.

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2023 – A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3
Patchliner Publication in PLoS Biol (2023) Authors: Hackney C., Flórez Salcedo P., Mueller E., Lund Koch T., Kjelgaard L., Watkins M., Zachariassen L., Sønderby Tuelung P., McArthur J., Adams D., Kristensen A., Olivera B., Finol-Urdaneta R., Safavi-Hemami H., Preben Morth J., Ellgaard L.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Application Note PDF
NaV, KV, GABAA – “Stem cell derived neurons recorded on Nanion’s Patchliner”
Patchliner application note: hiPS cell-derived neurons (iCell® Neurons) kindly provided by Cellular Dynamics International

Stem cell–derived neurons provide a novel and unique model for studying human drug targets in their physiologically relevant environment of terminally differentiated, postmitotic cells. It has been increasingly recognized that associated proteins modulate the physiology and pharmacology of neuronal proteins. Therefore, assays that investigate neuronal toxicity, drug effects, or basic cellular functions of neurons can mostly benefit from the development of human neurons derived from induced pluripotent stem cells (hiPSC-neurons). In 2011, Fujifilm Cellular Dynamics International (CDI) announced the commercial launch of human iCell® Neurons for use in neuroscience drug discovery, neurotoxicity screens, and other health research. This was the first commercially available iPSC-derived neuronal type available3. It was a mixture of GABAergic and glutamatergic post-mitotic neurons which rapidly regenerate post-thaw. Over the years, Fujifilm Cellular Dynamics International has developed a range of hiPSC-neurons with either healthy or diseased models. These can be used to study a number of different neurodegenerative diseases including Alzheimer’s Disease, one of the leading causes on death in the United States5.

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Kv7.2-7.3- “Cross-site study of neuronal KV7.2/7.3 heteromers on the Patchliner”
Patchliner application note: in collaboration with ApconiX

The KV7 channels are a family of voltage-gated potassium ion channels with five members (KV7.1 - 7.5) encoded by the KCNQ1-5 genes1. The channels exist as tetramers, with each subunit containing six transmembrane domains with cytoplasmic N- and C-termini. The long intracellular terminus is essential for tetramerization as well as interaction with critical regulators such as PIP2, calmodulin, protein kinase C and ankyrin G2. KV7-mediated currents are voltage activated, slowly activating and non-inactivating and are involved in repolarization of the cell membrane potential, thereby controlling cell excitability. KV7.1 channels are primarily expressed in cardiac cells whereas KV7.2, KV7.3 and KV7.5 are widely distributed in neuronal and primary sensory cells2. KV7.2/7.3 heteromeric channels primarily underlie the neuronal M-current (IKM)which plays a crucial role in repolarizing neuronal membrane potential after a depolarizing input which limits repetitive firing and is, therefore, a key mechanism in spike frequency adaptation.

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2023 – Development of automated patch clamp assays to overcome the burden of variants of uncertain significance in inheritable arrhythmia syndromes
Patchliner and SyncroPatch 384 Publication (Review) in Front. Physiol. (2023) Authors: Ma J.G., Vandenberg J.I., Ng C-A.

Advances in next-generation sequencing have been exceptionally valuable for identifying variants in medically actionable genes. However, for most missense variants there is insufficient evidence to permit definitive classification of variants as benign or pathogenic. To overcome the deluge of Variants of Uncertain Significance, there is an urgent need for high throughput functional assays to assist with the classification of variants. Advances in parallel planar patch clamp technologies has enabled the development of automated high throughput platforms capable of increasing throughput 10- to 100-fold compared to manual patch clamp methods. Automated patch clamp electrophysiology is poised to revolutionize the field of functional genomics for inheritable cardiac ion channelopathies. In this review, we outline i) the evolution of patch clamping, ii) the development of high-throughput automated patch clamp assays to assess cardiac ion channel variants, iii) clinical application of these assays and iv) where the field is heading.

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2023 – Recombinant cellular model system for human muscle‑type nicotinic acetylcholine receptor α1(2)β1δε
Patchliner and Buffer Solution Publication in Cell Stress and Chaperones (2023) Authors: Brockmöller S., Seeger T., Worek F., Rothmiller S.

The human muscle-type nicotinic acetylcholine receptor α12β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development.

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2023 – Baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating ATP-sensitive potassium channels
Patchliner Publication in Acta Pharmacologica Sinica (2023) Authors: Kong D., Du L., Liu R., Yuan T., Wang S., Wang Y., Lu Y., Fang L., Du G.

Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson’s disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 μM and 6.159 μM, respectively. K-ATP channel blockers glibenclamide (50 μM) or 5-hydroxydecanoate (5-HD, 250 μM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg−1·d−1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 μM) than glibenclamide (KD = 24.32 μM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.

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2023 – Pharmacology of P2X3and P2X2/3receptors in cell lines and hiPSC-derived neurons: An automated patch clamp study
SyncroPatch 384, Patchliner, Port-a-Patch Poster
Poster PDF
2023 – Functional characterization of human GAT-1 using solid supported membrane
SURFE2R N1 and Patchliner Poster
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2023 – An integrated approach for early in vitro seizure prediction utilizing hiPSC neurons and human ion channel assays
Patchliner Publication in Toxicological Sciences (2023) Authors: Rockley K., Roberts R., Jennings H., Jones K., Davis M., Levesque P., Morton M.

Seizure liability remains a significant cause of attrition throughout drug development. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. We assessed the activity of 15 pro-seizurogenic compounds (7 CNS active therapies, 4 GABA receptor antagonists, and 4 other reported seizurogenic compounds) using automated electrophysiology against a panel of 14 ion channels (Nav1.1, Nav1.2, Nav1.6, Kv7.2/7.3, Kv7.3/7.5, Kv1.1, Kv4.2, KCa4.1, Kv2.1, Kv3.1, KCa1.1, GABA α1β2γ2, nicotinic α4β2, NMDA 1/2A). These were selected based on linkage to seizure in genetic/pharmacological studies. Fourteen compounds demonstrated at least one “hit” against the seizure panel and 11 compounds inhibited 2 or more ion channels. Next, we assessed the impact of the 15 compounds on electrical signaling using human-induced pluripotent stem cell neurons in microelectrode array (MEA). The CNS active therapies (amoxapine, bupropion, chlorpromazine, clozapine, diphenhydramine, paroxetine, quetiapine) all caused characteristic changes to electrical activity in key parameters indicative of seizure such as network burst frequency and duration. The GABA antagonist picrotoxin increased all parameters, but the antibiotics amoxicillin and enoxacin only showed minimal changes. Acetaminophen, included as a negative control, caused no changes in any of the parameters assessed. Overall, pro-seizurogenic compounds showed a distinct fingerprint in the ion channel/MEA panel. These studies highlight the potential utility of an integrated in vitro approach for early seizure prediction to provide mechanistic information and to support optimal drug design in early development, saving time and resources.

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2023 – SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics
Patchliner Publication in Biological Research (2023) Authors: Prieto-Villalobos J., Lucero C.M., Rovegno M., Gómez G.I., Retamal M.A. Orellana J.A.

Background

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels.

Results

We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP.

Conclusions

We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.

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2023 – The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells
Patchliner and SyncroPatch 384 Pre-Print Publication in Preprints (2023) Authors: Petkova-Kirova P., Murciano N., Jansen J., Simionato G., Iacono G., Rotordam M.G., John T., Qiao M., Hertz L., Hoogendijk A.J., Becker N., Wagner C., von Lindern M., Egee S., Van den Akker E., Kaestner L.

(1) Background: The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood.

(2) Methods: Here we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch-clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. Besides this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties.

(3) Results: Both, Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single digit range, can be detected by transcriptome analysis of reticulocytes. Proteomics analysis of RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch-clamp method. While for the Gárdos channel the activity is higher in reticulocytes compared to mature RBCs, for Piezo1 the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch-clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs.

(4) Conclusions: We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

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2023 – Structure-based development and preclinical evaluation of the SARS-CoV-2 3C-like protease inhibitor simnotrelvir
Patchliner Publication in Nature Communications (2023) Authors: Jiang X., Su H., Shang W., Zhou F., Zhang Y., Zhao W, Zhang Q., Xie H., Jiang L., Nie T., Yang F., Xiong M., Huang X., Li M., Chen P., Peng S., Xiao G., Jiang H., Tang R., Zhang L., Shen J., Xu Y.

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CLpro) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CLpro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CLpro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

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2023 – Unravelling Novel SCN5A Mutations Linked to Brugada Syndrome: Functional, Structural, and Genetic Insights
Patchliner Publication in Int. J. Mol. Sci. (2023) Authors: Frosio A., Micaglio E., Polsinelli I., Calamaio S., Melgari D., Prevostini R., Ghiroldi A., Binda A., Carrera P., Villa M., Mastrocinque F., Presi S., Salerno R., Boccellino A., Anastasia L., Ciconte G.,Ricagno S., Pappone C., Rivolta I.

Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia causing potentially fatal ventricular tachycardia or fibrillation, mainly occurring during rest or sleep in young individuals without heart structural issues. It increases the risk of sudden cardiac death, and its characteristic feature is an abnormal ST segment elevation on the ECG. While BrS has diverse genetic origins, a subset of cases can be conducted to mutations in the SCN5A gene, which encodes for the Nav1.5 sodium channel. Our study focused on three novel SCN5A mutations (p.A344S, p.N347K, and p.D349N) found in unrelated BrS families. Using patch clamp experiments, we found that these mutations disrupted sodium currents: p.A344S reduced current density, while p.N347K and p.D349N completely abolished it, leading to altered voltage dependence and inactivation kinetics when co-expressed with normal channels. We also explored the effects of mexiletine treatment, which can modulate ion channel function. Interestingly, the p.N347K and p.D349N mutations responded well to the treatment, rescuing the current density, while p.A344S showed a limited response. Structural analysis revealed these mutations were positioned in key regions of the channel, impacting its stability and function. This research deepens our understanding of BrS by uncovering the complex relationship between genetic mutations, ion channel behavior, and potential therapeutic interventions.

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2023 – Evidence of Cytolysin A nanopore incorporation in mammalian cells assessed by a graphical user interface
Patchliner Publication in Nanoscale (2023) Authors: Lucas F.L.R., Finol-Urdaneta R.K., Van Thillo T., McArthur J.R., van der Heide N.J., Maglia G., Dedecker P., Strauss O., Wloka C.

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensor sallow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology. In CHO-K1 cells, SBPs modify the ionic current flowing though ClyA nanopores, thus demonstrating its potential for metabolite sensing of living cells. Moreover, we developed a graphical user interface for the analysis of the complex signals resulting from multiplexed APC recordings. This system lays the foundation to bridge the gap between recent advances in the nanopore field (e.g., proteomic and transcriptomic) and potential cellular applications.

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2023 – Target-based discovery of a broad spectrum flukicide
Patchliner Publication Pre-print in bioRxiv (2023) Authors: Sprague D.J., Park S-K., Gramberg S., Bauer L., Rohr C.M., Chulkov E.G., Smith E., Scampavia L., Spicer T.P., Haeberlein S., Marchant J.S.

Diseases caused by parasitic flatworms impart a considerable healthcare burden worldwide. Many of these diseases – for example, the parasitic blood fluke infection, schistosomiasis – are treated with the drug praziquantel (PZQ). However, PZQ is ineffective against disease caused by liver flukes from the genus Fasciola. This is due to a single amino acid change within the target of PZQ, a transient receptor potential ion channel (TRPMPZQ), in Fasciola species. Here we identify benzamidoquinazolinone analogs that are active against Fasciola TRPMPZQ. Structure-activity studies define an optimized ligand (BZQ) that caused protracted paralysis and damage to the protective tegument of these liver flukes. BZQ also retained activity against Schistosoma mansoni comparable to PZQ and was active against TRPMPZQ orthologs in all profiled species of parasitic fluke. This broad spectrum activity was manifest as BZQ adopts a pose within the binding pocket of TRPMPZQ dependent on a ubiquitously conserved residue. BZQ therefore acts as a universal activator of trematode TRPMPZQ and a first-in-class, broad spectrum flukicide.

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2023 – Nicotine-mediated effects in neuronal and mouse models of synucleinopathy
Patchliner Publication in Front. Neurosci. (2023) Authors: Fares M.B., Alijevic O., Johne S., Overk C., Hashimoto M., Kondylis A., Adame A., Dulize R., Peric D., Nury C., Battey J., Guedj E., Sierro N., Mc Hugh D., Rockenstein E., Kim C. Rissman R.A., Hoeng J., Peitsch M.C., Masliah E., Mathis C.

Introduction: Alpha-synuclein (α-Syn) aggregation, transmission, and contribution to neurotoxicity represent central mechanisms underlying Parkinson’s disease. The plant alkaloid “nicotine” was reported to attenuate α-Syn aggregation in different models, but its precise mode of action remains unclear.

Methods: In this study, we investigated the effect of 2-week chronic nicotine treatment on α-Syn aggregation, neuroinflammation, neurodegeneration, and motor deficits in D-line α-Syn transgenic mice. We also established a novel humanized neuronal model of α-Syn aggregation and toxicity based on treatment of dopaminergic neurons derived from human induced pluripotent stem cells (iPSC) with α-Syn preformed fibrils (PFF) and applied this model to investigate the effects of nicotine and other compounds and their modes of action.

Results and discussion: Overall, our results showed that nicotine attenuated α-Syn-provoked neuropathology in both models. Moreover, when investigating the role of nicotinic acetylcholine receptor (nAChR) signaling in nicotine’s neuroprotective effects in iPSC-derived dopaminergic neurons, we observed that while α4-specific antagonists reduced the nicotine-induced calcium response, α4 agonists (e.g., AZD1446 and anatabine) mediated similar neuroprotective responses against α-Syn PFF-provoked neurodegeneration. Our results show that nicotine attenuates α-Syn-provoked neuropathology in vivo and in a humanized neuronal model of synucleinopathy and that activation of α4β2 nicotinic receptors might mediate these neuroprotective effects.

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2023 – Brevetoxin versus Brevenal Modulation of Human Nav1 Channels
Patchliner Publication in Mar. Drugs (2023) Authors: Finol-Urdaneta R., Zhorov B., Baden D., Adams D.

Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-clamp. While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is >1000-fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels’ interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.

Read more about the publication here.

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2023 – Structure-activity relationship of dihydropyridines for rhabdomyosarcoma
Patchliner Publication in Biochem. & Biophys. Res. Comms. (2023) Authors: Chauhan S., Woods A.D., Bharathy N., Lian X., Ricker C.A., Mantz A., Zuercher W.J., Price L.H., Morton M.J., Durrant E., Corbel S.Y., Sampath S.C., Sampath S.C., Joslin J., Keller C.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

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2023 – The Putative Role of the Transient Receptor Potential Ion Channel of Vanilloid Type 2 in Red Blood Cell Storage Lesions
Patchliner and SyncroPatch 384 Publication in Transfus. Med. Hemother. (2023) Authors: Murciano N., Kaestner L.

Recently, a review about big data and artificial intelligence was published in “Transfusion Medicine and Hemotherapy,” highlighting the importance and chances of quality control of stored red blood cells (RBCs). RBC quality is decreased over storage time in a donor-dependent manner. Here, we want to emphasize that besides quality control, one has to further think about improving the RBC quality during storage, i.e., addressing storage lesions. A component of the storage lesion is the dissipation of the cation gradients across the RBC membrane, i.e., K+ will leak out of the RBC and Na+ enters the cell. So far, the molecular cause of the cation gradient dissipation remains elusive. To this end, we like to present a hypothesis for the involvement of the transient receptor potential channel of vanilloid type 2 (TRPV2).

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2023 – Pain-causing stinging nettle toxins target TMEM233 to modulate NaV1.7 function
Patchliner Publication in Nature Communications (2023) Authors: Jami S., Deuis J., Klasfauseweh T., Cheng X., Kurdyukov S., Chung F., Okorokov A., Li S., Zhang J., Cristofori-Armstrong B., Israel M., Ju R., Robinson S., Zhao P., Ragnarsson L., Andersson Å., Tran P., Schendel V., McMahon K., Tran H., Chin Y., Zhu Y., Liu J., Crawford T., Purushothamvasan S., Habib A., Andersson D., Rash L., Wood J., Zhao J., Stehbens S., Mobli M., Leffler A., Jiang D., Cox J., Waxman S., Dib-Hajj S., Neely G., Durek T., Vetter I.

Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.

Read more in the publication here.

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2023 – Assay for evaluation of proarrhythmic effects of herbal products: Case study with 12 Evodia preparations
Patchliner and SyncroPatch 384 Publication in Toxicology Reports (2023) Authors: Baltov B., Beyl S., Baburin I., Reinhardt J., Szkokan P., Garifulina A., Timin E., Kraushaar U., Potterat O., Hamburger M., Kügler P., Hering S.

Guidelines for preclinical drug development reduce the occurrence of arrhythmia-related side effects. Besides ample evidence for the presence of arrhythmogenic substances in plants, there is no consensus on a research strategy for the evaluation of proarrhythmic effects of herbal products. Here, we propose a cardiac safety assay for the detection of proarrhythmic effects of plant extracts based on the experimental approaches described in the Comprehensive In vitro Proarrhythmia Assay (CiPA). Microelectrode array studies (MEAs) and voltage sensing optical technique on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were combined with ionic current measurements in mammalian cell lines, In-silico simulations of cardiac action potentials (APs) and statistic regression analysis. Proarrhythmic effects of 12 Evodia preparations, containing different amounts of the hERG inhibitors dehydroevodiamine (DHE) and hortiamine were analysed. Extracts produced different prolongation of the AP, occurrence of early after depolarisations and triangulation of the AP in hiPSC-CMs depending on the contents of the hERG inhibitors. DHE and hortiamine dose-dependently prolonged the field potential duration in hiPSC-CMs studied with MEAs. In-silico simulations of ventricular AP support a scenario where proarrhythmic effects of Evodia extracts are predominantly caused by the content of the selective hERG inhibitors. Statistic regression analysis revealed a high torsadogenic risk for both compounds that was comparable to drugs assigned to the high-risk category in a CiPA study.

Read more in the publication here.

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2023 – Nonclinical Cardiovascular Assessment of the Soluble Guanylate Cyclase Stimulator Vericiguat
Patchliner Publication in JPET (2023) Authors: Himmel H., Lagrutta A., Vömel M., Amin R.P., Imredy J.P., Johnson T., Vinzing M., Prescott J., Blaustein R.O.

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action–related dose-dependent changes in systolic arterial blood pressure (up to −20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (−6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1.

SIGNIFICANCE STATEMENT There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.

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2023 – Antidepressant-like activity of a brain penetrant HCN channel inhibitor in mice
Patchliner Publication in Front. Pharmacol. (2023) Authors: Pinares-Garcia P., Spyrou J., McKenzie C.E., Forster I.C., Soh M.S., Syazwan E.M., Atif M. Reid C.A.

Changes in Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel function have been linked to depressive-like traits, making them potential drug targets. However, there is currently no peer-reviewed data supporting the use of a small molecule modulator of HCN channels in depression treatment. Org 34167, a benzisoxazole derivative, has been patented for the treatment of depression and progressed to Phase I trials. In the current study, we analysed the biophysical effects of Org 34167 on HCN channels in stably transfected human embryonic kidney 293 (HEK293) cells and mouse layer V neurons using patch-clamp electrophysiology, and we utilised three high-throughput screens for depressive-like behaviour to assess the activity of Org 34167 in mice. The impact of Org 34167 on locomotion and coordination were measured by performing rotarod and ledged beam tests. Org 34167 is a broad-spectrum inhibitor of HCN channels, slowing activation and causing a hyperpolarising shift in voltage-dependence of activation. It also reduced Ih-mediated sag in mouse neurons. Org 34167 (0.5 mg/kg) reduced marble burying and increased the time spent mobile in the Porsolt swim and tail suspension tests in both male and female BALB/c mice, suggesting reduced depressive-like behaviour. Although no adverse effects were seen at 0.5 mg/kg, an increase in dose to 1 mg/kg resulted in visible tremors and impaired locomotion and coordination. These data support the premise that HCN channels are valid targets for anti-depressive drugs albeit with a narrow therapeutic index. Drugs with higher HCN subtype selectivity are needed to establish if a wider therapeutic window can be obtained.

Flyer PDF
Investigating PIEZO1 using the Patchliner
Publication Link
2023 – KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells
Patchliner Publication in Biomolecules (2023) Authors: Chang C.W.T., Poudyal N., Verdugo D.A., Peña F., Stehberg J., Retamal M.A.

Background: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. Methods: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). Results: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. Results: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors.

2023 – Human Induced Pluripotent Stem Cell Derived Nociceptors suitable for Automated Patch Clamp High Throughput Pain Drug Discovery
Application Note PDF
Nav, Cav, Kv – High throughput APC recordings from RealDRGTM iPSC sensory neurons for analgesia drug discovery
Patchliner and SyncroPatch 384 application note: RealDRGs kindly provided by Anatomic Inc.
Conference poster
2023- Pharmacology of transient receptor potential cation (TRP) channels using different activation stimuli
Poster presented at the 7th RSC-BMCS / SCI Symposium on Ion Channels as Therapeutic Targets
Publication link
2023 – Automated Patch-Clamp and Induced Pluripotent Stem Cell-Derived Cardiomyocytes: A Synergistic Approach in the Study of Brugada Syndrome
Patchliner and SyncroPatch 384 Publication (review) in Int. J. Mol. Sci (2023) Authors: Melgari D., Calamaio S., Frosio A., Prevostini R., Anastasia L., Pappone C., Rivolta I.

The development of high-throughput automated patch-clamp technology is a recent breakthrough in the field of Brugada syndrome research. Brugada syndrome is a heart disorder marked by abnormal electrocardiographic readings and an elevated risk of sudden cardiac death due to arrhythmias. Various experimental models, developed either in animals, cell lines, human tissue or computational simulation, play a crucial role in advancing our understanding of this condition, and developing effective treatments. In the perspective of the pathophysiological role of ion channels and their pharmacology, automated patch-clamp involves a robotic system that enables the simultaneous recording of electrical activity from multiple single cells at once, greatly improving the speed and efficiency of data collection. By combining this approach with the use of patient-derived cardiomyocytes, researchers are gaining a more comprehensive view of the underlying mechanisms of heart disease. This has led to the development of more effective treatments for those affected by cardiovascular conditions.

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2023 – Beyond CBD: Inhibitory effects of lesser studied phytocannabinoids on human voltage-gated sodium channels
Patchliner Publication in Front. Physiol. (2023) Authors: Milligan C.J., Anderson L.L., McGregor I.S., Arnold J.C., Petrou S.

Introduction: Cannabis contains cannabidiol (CBD), the main non-psychoactive phytocannabinoid, but also many other phytocannabinoids that have therapeutic potential in the treatment of epilepsy. Indeed, the phytocannabinoids cannabigerolic acid (CBGA), cannabidivarinic acid (CBDVA), cannabichromenic acid (CBCA) and cannabichromene (CBC) have recently been shown to have anti-convulsant effects in a mouse model of Dravet syndrome (DS), an intractable form of epilepsy. Recent studies demonstrate that CBD inhibits voltage-gated sodium channel function, however, whether these other anti-convulsant phytocannabinoids affect these classic epilepsy drug-targets is unknown. Voltage-gated sodium (NaV) channels play a pivotal role in initiation and propagation of the neuronal action potential and NaV1.1, NaV1.2, NaV1.6 and NaV1.7 are associated with the intractable epilepsies and pain conditions.

Methods: In this study, using automated-planar patch-clamp technology, we assessed the profile of the phytocannabinoids CBGA, CBDVA, cannabigerol (CBG), CBCA and CBC against these human voltage-gated sodium channels subtypes expressed in mammalian cells and compared the effects to CBD.

Results: CBD and CBGA inhibited peak current amplitude in the low micromolar range in a concentration-dependent manner, while CBG, CBCA and CBC revealed only modest inhibition for this subset of sodium channels. CBDVA inhibited NaV1.6 peak currents in the low micromolar range in a concentration-dependent fashion, while only exhibiting modest inhibitory effects on NaV1.1, NaV1.2, and NaV1.7 channels. CBD and CBGA non-selectively inhibited all channel subtypes examined, whereas CBDVA was selective for NaV1.6. In addition, to better understand the mechanism of this inhibition, we examined the biophysical properties of these channels in the presence of each cannabinoid. CBD reduced NaV1.1 and NaV1.7 channel availability by modulating the voltage-dependence of steady-state fast inactivation (SSFI, V0.5 inact), and for NaV1.7 channel conductance was reduced. CBGA also reduced NaV1.1 and NaV1.7 channel availability by shifting the voltage-dependence of activation (V0.5 act) to a more depolarized potential, and for NaV1.7 SSFI was shifted to a more hyperpolarized potential. CBDVA reduced channel availability by modifying conductance, SSFI and recovery from SSFI for all four channels, except for NaV1.2, where V0.5 inact was unaffected.

Discussion: Collectively, these data advance our understanding of the molecular actions of lesser studied phytocannabinoids on voltage-gated sodium channel proteins.

Poster PDF
2018 – Expression and pharmacology of GluA2-containing AMPA receptors in cell lines and stem cell-derived neurons
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, Europhysiology Meeting 2018
Poster PDF
2015 – The backstage pass to study your favorite TRP channel
Port-a-Patch, Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) poster, TRP Meeting 2015
Poster PDF
2021 – Activation and inhibition of assay-ready TRPA1 and TRPV cells: an automated patch clamp study
Port-a-Patch, Patchliner and SyncroPatch 384i (a predecessor model of SyncroPatch 384), Biophysical Society Meeting 2021
Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Dr. Sonja Stölzle-Feix
CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96;
HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Webinar
12.09.2018 | Webinar: CiPA study: Bridging ion channel and myocyte data
Authors: Tim Strassmaier

Get up-to-date with the CiPA progress of the Myocyte and Ion Channel Work Goups: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines.

Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed.Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values.

With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.

Poster PDF
2015 – Complementary automated patch clamp, extracellular field potential and impedance recordings of iPSCs: safety screening tool box for the future
Webinar
2018 – HTS Phase I study: an update on progress of the CiPA Ion Channel Work Stream using the SyncroPatch 384PE and Patchliner
Presenter: Tim Strassmaier, Nanion Technologies Inc. USA, Source: Webinar: "CiPA study: Bridging ion channel and myocyte data", September 12, 2018
Title: CiPA myocyte phase II validation study results: cross-site comparison using the CardioExcyte 96

The CiPA HTS Ion Channel Working Group finalized its phase I study in 2017. Amongst other external sites, Nanion Technologies in Germany, USA and Japan participated with the Patchliner and the SyncroPatch 384PE in this study. A comparative view of the ion channel targets and a cross-platform and cross-site comparison will be presented. Furthermore, results from the myocyte Work Stream using arrhythmogenic compounds will be compared and confirmed with patch clamp data derived from the HTS Work Stream.

Please note: The original webinar presentation contained 8 slides with data of an upcoming publication. Due to confidentiality reasons, the relevant slides were cut out of the movie.

Poster PDF
2018 – Combining electrophysiology and contractility recordings for more complete assessment of hiPSC-CMs
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384), Patchliner and CardioExcyte 96 poster, Europhysiology Meeting 2018
Webinar
28.07.2015 | Webinar: High Throughput and High Fidelity: Automated Patch Clamp in Screening and Research
Authors: Alfred L. George; Dr. Carlos Vanoye,
User meeting video
14.10.2020 | Webinar: Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms.
Authors: Marc Rogers, This is an on-demand webinar from Nan]i[on and Friends 2020.

Whilst voltage-gated ion channels formed the bulk of academic and industrial effort in developing and utilising APC assays for ion channel drug discovery, recent years have seen increasing interest in ligand-gated receptors. These targets offer specific challenges for APC systems in terms of lower channel expression, rapid application and wash-off of ligands, and loss of responsiveness due to short- and long-term desensitisation. In this presentation I will outline successful development of pipette- and tip-based APC assay formats for the rapidly-activating ASIC1A channel on the Patchliner and SyncroPatch384i.

User meeting video
15.10.2020 | Webinar: Benchmarking best practices and calibration standards for HTS hERG recordings for improved proarrhythmic assessment
Dr. Alison Obergrussberger (Nanion Technologies), This is an on-demand webinar from Nan]i[on and Friends 2020.

The use of automated patch clamp (APC) electrophysiology in cardiac safety screening has increased over the years, and APC is now an established and accepted technique in most, if not all, safety testing laboratories. Since the introduction of the ICH S7B non-clinical guidance in November 2005 which requires all new drugs to be tested for activity on the IKr current carried by hERG expressed in recombinant cell lines using the patch-clamp technique, very few drugs have been withdrawn from the market due to pro-arrhythmic complications. APC has become the major workhorse in safety testing laboratories and is now considered to be the gold standard. Furthermore, with the introduction of the comprehensive in vitro pro-arrhythmia assay (CiPA) which recommends expanding electrophysiological recordings to include other cardiac ion channels, APC will continue to play a major role in cardiac safety testing. Recently, a large study comparing the results of a set of standard compounds tested on different instruments at different sites has been published[1] which highlights the need for standardized protocols for reliable results, for example, for hERG recordings.

We have undertaken a study to identify key parameters that can affect IC50 values of compounds acting on hERG using the medium and high throughput APC systems, Patchliner, SyncroPatch 384PE and SyncroPatch 384i. Effects of experimental parameters such as voltage protocol, incubation time, labware, compound storage time and replicate number on IC50 values of a set of CiPA compounds will be presented and recommendations for best practices for hERG measurements using APC is provided. Furthermore, as outlined in the 2020 Best Practice Consideration for In vitro Studies, ‘The concentration of compound to which the cells were exposed should be verified by applying a validated analytical method to the solution collected from the cell chamber’[2] in patch clamp studies. Nanion has implemented a new procedure that enables sample collection from used wells from the NPC-384 chips and this will be described.

Poster PDF
2022 – Characterization of ion channel currents endogenously expressed in Neuro2A cells using automated patch clamp
Poster PDF
2022 – The role of LRRC8 in the hypotonic stress response of human keratinocytes
Poster PDF
2020 – Kinetic and pharmacological properties of P2X3 and P2X2/3 receptors
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384 instrument) and Patchliner poster, 64th Annual Meeting of the Biophysical Society
Poster PDF
2021 – Development and validation of ASIC1a ligand-gated ion channel drug discovery assays on automated patch clamp platforms
Poster PDF
2020 – Reliable Identification of hERG Liability in Drug Discovery by Automated Patch Clamp
SyncroPatch 384i (a predecessor model of SyncroPatch 384) and Patchliner poster, 64th Annual Meeting of the Biophysical Society
Poster PDF
2018 – Investigating pain pathways by inhibition of voltage-gated sodium channels
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner poster, FENS Meeting 2018
Poster PDF
2017 – Cardiomyocytes in Voltage Clamp and Current Clamp by Automated Patch Clamp
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner poster, BPS Meeting 2017
Poster PDF
2021 – Reliable identification of cardiac liability in drug discovery using automated patch clamp: Considerations and best practices for high throughput recordings of NaV 1.5
Patchliner and SyncroPatch 384i (a predecessor model of SyncroPatch 384), Physiology 202
Application Note PDF
VRAC – “The role of LRRC8 in the hypotonic stress response of human keratinocytes”
SyncroPatch 384 and Patchliner application note:    

The human skin is constantly exposed to various stress factors such as temperature changes, mechanical stress, different humidity levels, air pollution or radiation. These factors can have a tremendous impact on the skin and can contribute to barrier disruption and inflammation, dry and fragile skin as well as premature ageing. Recent advances in different research areas point to an important role of LRRC8 volume regulated anion channels (VRACs) in a plethora of different physiological processes. The function of LRRC8 has been characterized in human keratinocytes and in the native human epidermis and the LRRC8 ion channel has been proposed to be a novel molecular target to modulate keratinocyte differentiation in a recent patent.LRRC8A (also named SWELL1) has been identified as the first essential component of VRACs in various cell types. LRRC8A is composed of four transmembrane domains and a C-terminal domain containing up to 17 leucine-rich repeats. Together with four additional LRRC8 family members (LRRC8B-E) it assembles into hetero-hexameric complexes. Interestingly, the LRRC8 subunit composition differs between cell types and influences VRAC properties such as inactivation kinetics, voltage-dependence and selectivity of the transported osmolyte. The generation of LRRC8A-/- knockout HaCaT keratinocytes have provided evidence for the essential function of LRRC8A in hypotonic stress response of human keratinocytes.In this Application Note we show electrophysiological data from WT and LRRC8A-/- knockout HaCaT keratinocytes which corroborate the essential function of LRRC8A in keratinocytes.

Webinar
07.09.2021 | Webinar: Automated patch clamp assay development for the study of red blood cells (RBCs) in health and disease
Presenters: Nicoletta Murciano (PhD Candidate - Nanion Technologies ), Dr. Lars Kaestner (Professor - Saarland University)

Calcium (Ca2+) is a universal signalling molecule and is critically important in regulating many physiological functions and survival of RBCs. Amongst others, intracellular Ca2+ controls cell volume and deformability. This process plays a substantial role in RBCs since their volume needs to adapt when passing blood vessel constrictions during the flow. Excessive Ca2+ uptake also leads to accelerated cell clearance causing anaemia.

Therefore, studying Ca2+ regulation is crucial to understand RBC diseases. Piezo1, KCa3.1 (Gardos channel) and NMDA receptors are three channels present in the RBC membrane and critical for Ca2+ regulation.

We developed functional assays to measure these channels in healthy and diseased RBCs populations using electrophysiological tools, contributing to the characterization of RBC diseases.

User meeting video
14.10.2020 | Webinar: Measurement of Transient receptor potential cation (TRP) channels using the Patchliner and Port-a-Patch
Dr. András Horváth (Nanion Technologies; Germany) This is an on-demand webinar from Nan]i[on and Friends 2020.

CHO cells expressing transient receptor potential cation channel V (TRPV) members 1, 3 and 4 and subfamily M member 8 were studied using our automated patch clamp systems, the Patchliner Octo (PL) and Port-a-Patch Perfusion (PaPP). During the recordings, heat, cold and/or ligand activation was performed. A classical ramp pulse-protocol (–100 mV to 100 mV) was applied.

Heat activation of TRPV1, 3, 4 channels was performed repeatedly by the heated pipetted (37-45 °C) of the PL. Interestingly ruthenium red (RR, 50 and 200 µM) was not able to prevent heat activation. Experiments involving TRPV4 were also performed on the PaPP. The cannel could be activated by heat and only partially blocked by RR. Ligand activation could be also performed on the PL (10 µM Capsaicin – TRPV1, 200 µM 2-APB – TRPV3, 100 nM GSK1016790 – TRPV4) and TRPV4 on the PaPP. In all cases the effect could be inhibited using blockers. TRPM8 channel could be repetitively activated using solution at 10°C on the PaPP at 10 °C. Capsazepine (10 µM) was used to block the activated current.

Both the PL and PaPP are powerful tools to study TRP channel physiology (both using heat activation and ligand activation) and could be used to find compounds which block the temperature and ligand response separately.

Webinar
20.11.2018 | Webinar: The RELEVANCE of ion channel interplay – Voltage-activated channels in non-excitable cells
RELEVANCE is an EU funded innovative training network and investigates in five scientific work packages different aspects, were the characterisation of red blood cells has an societal importance, such as in transfusion medicine, anaemias, diagnostics or in sports medicine. This will unavoidably result in the investigation of ion channels. The webinar is a joined presentation by a principle investigator of RELEVANCE, Prof. Lars Kaestner (Saarland University) and an early stage researcher, Maria Giustina Rotordam (Nanion Technologies, Munich).

Piezo1, KCa3.1 (Gardos channel) and CaV2.1 are three channels present in the red blood cell membrane. We will highlight the role of these channels in Hereditary Xerocytosis as well as in the Gardos Channelopathy using electrophysiological tools. Since red blood cells are everything but under suspicion to be excitable cells, we will take these cells as an example to show that KCa3.1, CaV2.1 and Piezo1 present an intimate interplay providing evidence that voltage-activated channels can well play a substantial role in non-excitable cells.

Publication link
2012 – Natural and artificial ion channels for biosensing platforms
Port-a-Patch, Patchliner, SyncroPatch 96 ((a predecessor model of SyncroPatch 384PE) and Vesicle Prep Pro Publication in Analytical and Bioanalytical Chemistry (2012) Authors: Steller L., Kreir M., Salzer R.

The single-molecule selectivity and specificity of the binding process together with the expected intrinsic gain factor obtained when utilizing flow through a channel have attracted the attention of analytical chemists for two decades. Sensitive and selective ion channel biosensors for high-throughput screening are having an increasing impact on modern medical care, drug screening, environmental monitoring, food safety, and biowarefare control. Even virus antigens can be detected by ion channel biosensors. The study of ion channels and other transmembrane proteins is expected to lead to the development of new medications and therapies for a wide range of illnesses. From the first attempts to use membrane proteins as the receptive part of a sensor, ion channels have been engineered as chemical sensors. Several other types of peptidic or nonpeptidic channels have been investigated. Various gating mechanisms have been implemented in their pores. Three technical problems had to be solved to achieve practical biosensors based on ion channels: the fabrication of stable lipid bilayer membranes, the incorporation of a receptor into such a structure, and the marriage of the modified membrane to a transducer. The current status of these three areas of research, together with typical applications of ion-channel biosensors, are discussed in this review.

Publication link
2019 – Role of High‐Throughput Electrophysiology in Drug Discovery
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i), Patchliner and Port-a-Patch review article in Current Protocols in Pharmacology (2019) Authors: Liu C., Li T., Chen J.

Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high‐throughput electrophysiology and its applications in drug discovery is provided.

Publication link
2020 – Automated Patch Clamp in Drug Discovery: major breakthroughs and innovation in the last decade
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i instrument), Patchliner and Port-a-Patch Publication in Expert Opinion on Drug Discovery (2020) Authors: Obergrussberger A., Friis S., Brüggemann A., Fertig N.

Patch-clamp electrophysiology remains an important technique in studying ion channels; indeed, it is still considered the gold standard since it was first described by Neher and Sakmann in the 1970s [1]. Ion channels are integral membrane proteins which allow ion current flow across the cell membrane. They are involved in almost all physiological processes, and their malfunction underlies many disease states, making them important pharmacological targets. Conventional patch clamp is a very information-rich technique, but it requires skilled personnel to perform experiments, and typically, only one experiment can be performed at a time. In the late 1990s and early 2000s, the field of ion-channel research was revolutionized by the development of the automated patch-clamp (APC) technique. The most successful approach involved replacing the patch-clamp pipette with a planar substrate (for review, see [2]), making the experiments easier to perform and offering the option for recording multiple cells in parallel. In the last two decades, much has changed in the field of ion-channel drug discovery and APC, with increased throughput and enhanced simplicity. We summarize the main changes in the last decade and attempt to look into the future of what’s to come.

Publication link
2011 – State-of-the-art automated patch clamp devices: heat activation, action potentials, and high throughput in ion channel screening
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) Publication in Frontiers in Pharmacology (2011) Authors: Stoelzle S., Obergrussberger A., Brüggemann A., Haarmann C., George M., Kettenhofen R., Fertig N.

Ion channels are essential in a wide range of cellular functions and their malfunction underlies many disease states making them important targets in drug discovery. The availability of standardized cell lines expressing ion channels of interest lead to the development of diverse automated patch clamp (APC) systems with high-throughput capabilities. These systems are now available for drug screening, but there are limitations in the application range. However, further development of existing devices and introduction of new systems widen the range of possible experiments and increase throughput. The addition of well controlled and fast solution exchange, temperature control and the availability of the current clamp mode are required to analyze standard cell lines and excitable cells such as stem cell-derived cardiomyocytes in a more physiologically relevant environment. Here we describe two systems with different areas of applications that meet the needs of drug discovery researchers and basic researchers alike. The here utilized medium throughput APC device is a planar patch clamp system capable of recording up to eight cells simultaneously. Features such as temperature control and recordings in the current clamp mode are described here. Standard cell lines and excitable cells such as stem cell-derived cardiomyocytes have been used in the voltage clamp and current clamp modes with the view to finding new drug candidates and safety testing methods in a more physiologically relevant environment. The high-throughput system used here is a planar patch clamp screening platform capable of recording from 96 cells in parallel and offers a throughput of 5000 data points per day. Full dose response curves can be acquired from individual cells reducing the cost per data point. The data provided reveals the suitability and relevance of both APC platforms for drug discovery, ion channel research, and safety testing.

Publication link
2012 – HTS techniques for patch clamp-based ion channel screening – economy and advances
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) Publication in Expert Opinion on Drug Discovery (2012) Authors: Farre C. and Fertig N.

Introduction: Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. “Going planar” proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. Areas covered: This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Expert opinion: Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Publication link
2010 – Renaissance of ion channel research and drug discovery by patch clamp automation
Port-a-Patch, Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE)  Publication in Future Medical Chemistry (2010) Authors: Farre C. and Fertig N.

Ion channels are highly intriguing biophysical entities that play an incredibly subtle role in the concerted actions in which they are involved, and that also have a crucial impact on inter- and intra-cellular communication. They respond to numerous kinds of stimuli and play a decisive role in the vitality of all living organisms. Ion channels are involved in the function of the cardiovascular and nervous systems and their malfunction underlies numerous diseases and indications. For exactly these reasons, ion channels have for decades been, and are still, the subject of in-depth research into a very broad range of important therapeutic areas. As membrane-bound proteins they are highly ‘druggable’ targets, being readily accessible to small molecules that are capable of fine tuning ion channel function by pharmacological modulation. Approximately 15% of the most successful drugs target ion channels, although ion channels have traditionally been difficult to screen due to a lack of adequate assays. Many of the marketed ion channel drugs were actually not discovered in rational drug-discovery programs, but rather empirically and by serendipity since the available ion channel-screening techniques typically confer a tradeoff between high content and high throughput.

Publication link
2015 – Novel screening techniques for ion channel targeting drugs
Patchliner, SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) and CardioExcyte 96 Publication in Channels (2015) Authors: Obergrussberger A., Stölzle-Feix S., Becker N., Brüggemann A., Fertig N., Möller C.

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

User meeting video
15.10.2020 | Webinar: ICH S7B best practices considerations – New Q&As and Benchmarking best practices
Dr. Sonja Stoelzle-Feix (Nanion Technologies), This is an on-demand webinar from Nan]i[on and Friends 2020.

The ICH E14/S7B Implementation Working Group released a draft version on August 28th 2020 on “Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential Questions and Answers”. This document is open for public consultation and comprises proposed revisions for some sections of the current Q&A´s for ICH E14. Furthermore, new Q&A for ICH S7B are included.

This talk will focus on best practice outlines as depicted in the draft version, specifically on in vitro cardiac ion channel assays. The ultimate goal is to provide a more robust and reproducible evaluation of potency of drug block of cardiac ion channel current using patch clamp techniques and heterologous expression systems.

Webinar
28.07.2015 | Webinar: High Throughput and High Fidelity: Automated Patch Clamp in Screening and Research
The webinar covers the use of the Patchliner and the SyncroPatch 384/768PE for characterization of ion channels and screening of ion channel active compounds.

The webinar covers the use of the Patchliner and the SyncroPatch 384/768PE for characterization of ion channels and screening of ion channel active compounds.

Both automated patch clamp systems support high quality, giga-seal recordings, but differ in throughput capabilities and experimental features.
The Patchliner, introduced in 2006, records eight cells in parallel, and is a highly appreciated research platform in industry and academia alike. Experimental features include individual pulse protocols, automated current clamp recordings, internal solution exchange, rapid perfusion, temperature control, and temperature jumps. In addition, scarce and expensive cells can readily be used because of optimized cell catch procedures requiring less than 500 cells/ml.

The SyncroPatch 384/768PE , introduced in 2013, has been adopted by big pharma and academic core facilities around the globe. The SyncroPatch 384/768PE it fully compatible with high throughput screening standards and can be integrated into existing hardware and software. The system supports giga seal recording from up to 768 cells in parallel, excellent voltage-control of patch-clamped cells, fast solution exchange, and has been validated with a wide variety of ion channel targets. Today, the SyncroPatch 384/768PE is utilized for academic research and HTS ion channel drug discovery.

Publication link
2023 – Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384 instrument) and Patchliner Publication in Mol. Pharmaceutics (2023) Authors: Finol-Urdaneta R.K., McArthur J.R., Aboelela A., Bujaroski R.S., Majed H., Rangel A., Adams D.J., Ranson M., Kelso M.J., Buckley B.J.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 μM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 μM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Publication link
2022 – Development of ASIC1a ligand-gated ion channel drug screening assays across multiple automated patch clamp platforms
SyncroPatch 384 and Patchliner Publication in Frontiers in Molecular Neuroscience (2022) Authors: Ridley J., Manyweathers S., Tang R., Goetze T., Becker N., Rinke-Weiß I., Kirby R., Obergrussberger A., Rogers M.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

Publication link
2020 – Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment
Patchliner and SyncroPatch 384i (SyncroPatch 384PE a predecessor model) Publication in the Journal of Pharmacological and Toxicological Methods (2020) Authors: Brinkwirth N., Takasuna K., Masafumi D., Becker N., Obergrussberger A., Friis S., Furukawa Y., Hasegawa Y., Oka T., Ohtsuki A., Fertig N., Stoelzle-Feix S.

Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. Methods: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values.

Publication link
2021 – Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for NaV1.5 peak current in a high throughput screening environment
SyncroPatch 384 and  Patchliner Publication in Journal of Pharmacological and Toxicological Methods (2021) Authors: Rotordam M.G., Obergrussberger A., Brinkwirth N., Takasuna K., Becker N., Horvátha A., Goetze T.A., Rapedius M., Furukawa H., Hasegawa Y., Oka T., Fertig N., Stoelzle-Feix S

For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC50s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices.

Publication link
2020 – Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells
Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i instrument) Publication in Nature Scientific Reports (2020) Authors: Kramer J., Himmel H.M., Lindqvist A., Stoelzle-Feix S., Chaudhary K.W., Li D., Bohme G.A., Bridgland-Taylor M., Hebeisen S., Fan J., Renganathan M., Imredy J., Humphries E.S.A, Brinkwirth N., Strassmaier T., Ohtsuki A., Danker T., Vanoye C., Polonchuk L., Fermini B., Pierson J.B. & Gintant G.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug’s cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCaV1.2 [L-Type ICa], peak hNaV1.5, [Peak INa], late hNaV1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNaV1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNaV1.5 blocking drugs. hERG and hNaV1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Publication link
2020 – Reengineering an Antiarrhythmic Drug Using Patient hiPSC Cardiomyocytes to Improve Therapeutic Potential and Reduce Toxicity
Patchliner and SyncroPatch 768PE (a predecessor model of the SyncroPatch 768i instrument) Publication in Cell Stem Cell (2020) Authors: McKeithan W. L., Feyen D.A.M., Bruyneel A.A.N., Okolotowicz K.J., Ryan D.A., Sampson K.J., Potet F., Savchenko A., Gómez-Galeno J., Vu M., Serrano R., George Jr. A.L., Kass R.S., Cashman J.R., Mercola M.

Modeling cardiac disorders with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes is a new paradigm for preclinical testing of candidate therapeutics. However, disease-relevant physiological assays can be complex, and the use of hiPSC-cardiomyocyte models of congenital disease phenotypes for guiding large-scale screening and medicinal chemistry have not been shown. We report chemical refinement of the antiarrhythmic drug mexiletine via high-throughput screening of hiPSC-CMs derived from patients with the cardiac rhythm disorder long QT syndrome 3 (LQT3) carrying SCN5A sodium channel variants. Using iterative cycles of medicinal chemistry synthesis and testing, we identified drug analogs with increased potency and selectivity for inhibiting late sodium current across a panel of 7 LQT3 sodium channel variants and suppressing arrhythmic activity across multiple genetic and pharmacological hiPSC-CM models of LQT3 with diverse backgrounds. These mexiletine analogs can be exploited as mechanistic probes and for clinical development.

Publication link
2018 – An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery
SyncroPatch 384/768PE (a predecessor model of SyncroPatch 384/768i) and Patchliner Publication in Expert Opinion on Drug Discovery Authors: Obergrussberger A., Goetze T.A., Brinkwirth N., Becker N., Friis S., Rapedius M., Haarmann C., Rinke-Weiß I., Stölzle-Feix S., Brüggemann A., George M., Fertig N.

Introduction: Automated patch clamp (APC) devices have become commonplace in many industrial and academic labs. Their ease-of-use and flexibility have ensured that users can perform routine screening experiments and complex kinetic experiments on the same device without the need for months of training and experience. APC devices are being developed to increase throughput and flexibility. Areas covered: Experimental options such as temperature control, internal solution exchange and current clamp have been available on some APC devices for some time, and are being introduced on other devices. A comprehensive review of the literature pertaining to these features for the Patchliner, QPatch and Qube and data for these features for the SyncroPatch 384/768PE, is given. In addition, novel features such as dynamic clamp on the Patchliner and light stimulation of action potentials using channelrhodosin-2 is discussed. Expert opinion: APC devices will continue to play an important role in drug discovery. The instruments will be continually developed to meet the needs of HTS laboratories and for basic research. The use of stem cells and recordings in current clamp mode will increase, as will the development of complex addons such as dynamic clamp and optical stimulation on high throughput devices.

Publication link
2020 – A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm
Patchliner and SyncroPatch 384PE (a predecessor model of the SyncroPatch 384i instrument) Publication in Toxicology and Applied Pharmacology (2020) Authors: Ridder B.J., Leishman D.J., Bridgland-Taylor M., Samieegohar M., Han X, Wu W.W., Randolph A., Tran P., Sheng J., Danker T., Lindqvist A., Konrad D., Hebeisen S., Polonchuk L., Gissinger E., Renganathan M., Koci B., Wei H., Fan J., Levesque P., Kwagh J., Imredy J., Zhai J., Rogers M., Humphries E., Kirby R., Stoelzle-Feix S., Brinkwirth N., Rotordam M.G., Becker N., Friis S., Rapedius M., Goetze T.A., Strassmaier T., Okeyo G., Kramer J., Kuryshev Y., Wu C., Himmel H., Mirams G.R., Strauss D.G., Bardenet R., Li Z.

Highlights: A new regulatory paradigm promotes the integration of nonclinical and clinical data. Lack of uncertainty quantification hindered using hERG potency in the new paradigm. A systematic method was established to address this limitation. Analysis supports using different safety margin thresholds in different context. Abstract: - Introduction hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. - Methods A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian Hierarchical Modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. - Results A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. - Discussion This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

Publication link
2014 – Early identification of hERG liability in drug discovery programs by automated patch clamp
Patchliner and SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) Publication in Frontiers in Pharmacology (2014) Authors: Danker T., Moeller C.

Blockade of the cardiac ion channel coded by human ether-à-gogo-related gene (hERG) can lead to cardiac arrhythmia, which has become a major concern in drug discovery and development. Automated electrophysiological patch clamp allows assessment of hERG channel effects early in drug development to aid medicinal chemistry programs and has become routine in pharmaceutical companies. However, a number of potential sources of errors in setting up hERG channel assays by automated patch clamp can lead to misinterpretation of data or false effects being reported. This article describes protocols for automated electrophysiology screening of compound effects on the hERG channel current. Protocol details and the translation of criteria known from manual patch clamp experiments to automated patch clamp experiments to achieve good quality data are emphasized. Typical pitfalls and artifacts that may lead to misinterpretation of data are discussed. While this article focuses on hERG channel recordings using the QPatch (Sophion A/S, Copenhagen, Denmark) technology, many of the assay and protocol details given in this article can be transferred for setting up different ion channel assays by automated patch clamp and are similar on other planar patch clamp platforms.

Publication link
2017 – Automated Patch Clamp Recordings of Human Stem Cell- Derived Cardiomyocytes.
Patchliner and SyncroPatch 384PE (a predecessor model of SyncroPatch 384i) book chapter in Stem Cell-Derived Models in Toxicology (2017) Authors: Obergrussberger A., Haarmann C., Stölzle-Feix S., Becker N., OhtsukiA., Brüggemann A., George M., Fertig N.

Patch clamp remains the gold standard for studying ion channel activity within cell membranes. Conventional patch clamp is notoriously low throughput and technically demanding making it an unsuitable technique for high-throughput screening (HTS). Automated patch clamp (APC) devices have done much to increase throughput and improve ease of use, particularly when using standard cell line cells such as HEK and CHO. In recent years, however, the use of human-induced pluripotent stem cells (hiPSCs) has become increasingly important, especially for safety screening in response to the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative introduced in 2013. The goal of this initiative is to standardize assays, targets, and cell types. One part of the paradigm focuses on the use of APC and hiPSC cardiomyocytes. This chapter describes two automated patch clamp devices recording from up to 8 or 384 cells simultaneously using hiPSC cardiomyocytes. In the voltage clamp mode, voltage-gated Na+ (NaV), Ca2+ (CaV), and K+ (KV) channels could be recorded, and pharmacology using tetracaine, a NaV channel blocker, is described. Additionally, action potentials in the current clamp mode were recorded, and examples are shown including the effect of nifedipine, a CaV channel blocker. Detailed methods are provided for cell culture and harvesting of hiPSCs for use on APC devices. Protocols are also provided for voltage and current clamp recordings on the Patchliner, and voltage clamp experiments on the SyncroPatch 384PE APC instruments.

Publication link
2011 – Automated electrophysiology makes the pace for cardiac ion channel safety screening
Patchliner and SyncroPatch 96 (a predecessor model of SyncroPatch 384PE) Publication in Frontiers in Pharmacology (2011) Authors: Möller C., Witchel H.

The field of automated patch-clamp electrophysiology has emerged from the tension between the pharmaceutical industry’s need for high-throughput compound screening versus its need to be conservative due to regulatory requirements. On the one hand, hERG channel screening was increasingly requested for new chemical entities, as the correlation between blockade of the ion channel coded by hERG and torsades de pointes cardiac arrhythmia gained increasing attention. On the other hand, manual patch-clamping, typically quoted as the “gold-standard” for understanding ion channel function and modulation, was far too slow (and, consequently, too expensive) for keeping pace with the numbers of compounds submitted for hERG channel investigations from pharmaceutical R&D departments. In consequence it became more common for some pharmaceutical companies to outsource safety pharmacological investigations, with a focus on hERG channel interactions.This outsourcing has allowed those pharmaceutical companies to build up operational flexibility and greater independence from internal resources, and allowed them to obtain access to the latest technological developments that emerged in automated patch-clamp electrophysiology – much of which arose in specialized biotech companies. Assays for nearly all major cardiac ion channels are now available by automated patch-clamping using heterologous expression systems, and recently, automated action potential recordings from stem-cell derived cardiomyocytes have been demonstrated. Today, most of the large pharmaceutical companies have acquired automated electrophysiology robots and have established various automated cardiac ion channel safety screening assays on these, in addition to outsourcing parts of their needs for safety screening.

Publication link
2016 – Coupling Data Mining and Laboratory Experiments to Discover Drug Interactions Causing QT Prolongation
Patchliner Publication in Journal of the American College of Cardiology (2016) Authors: Lorberbaum T., Sampson K.J., Chang J.B., Iyer V., Woosley R.L., Kass R.S., Tatonetti N.P.

Background:QT interval-prolonging drug-drug interactions (QT-DDIs) may increase the risk of life-threatening arrhythmia. Despite guidelines for testing from regulatory agencies, these interactions are usually discovered after drugs are marketed and may go undiscovered for years.Objectives:Using a combination of adverse event reports, electronic health records (EHR), and laboratory experiments, the goal of this study was to develop a data-driven pipeline for discovering QT-DDIs.Methods:1.8 million adverse event reports were mined for signals indicating a QT-DDI. Using 1.6 million electrocardiogram results from 380,000 patients in our institutional EHR, these putative interactions were either refuted or corroborated. In the laboratory, we used patch-clamp electrophysiology to measure the human ether-à-go-go-related gene (hERG) channel block (the primary mechanism by which drugs prolong the QT interval) to evaluate our top candidate.Results:Both direct and indirect signals in the adverse event reports provided evidence that the combination of ceftriaxone (a cephalosporin antibiotic) and lansoprazole (a proton-pump inhibitor) will prolong the QT interval. In the EHR, we found that patients taking both ceftriaxone and lansoprazole had significantly longer QTc intervals (up to 12 ms in white men) and were 1.4 times more likely to have a QTc interval above 500 ms. In the laboratory, we found that, in combination and at clinically relevant concentrations, these drugs blocked the hERG channel. As a negative control, we evaluated the combination of lansoprazole and cefuroxime (another cephalosporin), which lacked evidence of an interaction in the adverse event reports. We found no significant effect of this pair in either the EHR or in the electrophysiology experiments. Class effect analyses suggested this interaction was specific to lansoprazole combined with ceftriaxone but not with other cephalosporins.Conclusions:Coupling data mining and laboratory experiments is an efficient method for identifying QT-DDIs. Combination therapy of ceftriaxone and lansoprazole is associated with increased risk of acquired long QT syndrome.

Publication link
2011 – Cardiac Toxicity
Patchliner and Port-a-Patch book chapter in ADMET for Medicinal Chemists: A Practical Guide (2011) Authors: Kettenhofen R., Schwengberg S.
Publication link
2014 – Automated Patch Clamp Analysis of nAChα7 and NaV1.7 Channels
Port-a-Patch and Patchliner Publication in Current Protocols in Pharmacology (2014) Authors: Obergrussberger A., Haarmann C., Rinke I., Becker N., Guinot D., Brueggemann A., Stoelzle-Feix S., George M., Fertig N.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (NaV1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of NaV1.7, and activation and allosteric modulation of nAChα7R.

Publication link
2010 – Cor.At Cardiomyocytes: Primary-like Cardiomyocytes for Manual and Automated Electrophysiological Screening
Port-a-Patch and Patchliner Publication in Lonza Resource Notes (2010) Authors: Kettenhofen R., Stölzle S.

Cor.At® Cardiomyocytes are derived from mouse embryonic stem cells (mESC). During differentiation of the mESC, about 5% of all cells develop into cardiomyocytes. Using transgenic mESC with the puromycin resistance cassette under the control of the cardiac α-myosin heavy chain-(MHC) promoter, 99.9% pure Cor.At® Cardiomyocytes can be selected from the large amount of noncardiac myocyte cell population by the application of puromycin. For long-term storage, Cor.At® Cells are deep frozen as single cell suspensions in liquid nitrogen or -150°C deep freezers. Quality control strategies are implemented to guarantee lot-to-lot reproducibility and uniformity of functional properties of Cor.At® Cardiomyocytes for a storage period of at least 12 months. Thawed Cor.At®  Cardiomyocytes readily form spontaneously and synchronously contracting monolayers overnight. Seeded in low density on cover slips, Cor.At® Cardiomyocytes can be applied to manual patch clamp for the recording of action potentials as well as all three typical cardiac ion currents INa, ICa,L and IK (data not shown). Additionally, single cell suspensions of pre-cultured  Cor.At® Cardiomyocytes can be readily analyzed with very high success rates in automated patch clamp systems like the Port-a-Patch® and Patchliner® from Nanion Technologies GmbH, Munich, Germany, as well as in other automated patch clamp systems (data not shown). The uniqueness of both Nanion systems is their capability to record action potentials in the current clamp mode and the possibility to perform the recordings at physiological temperature in addition to the standard measurements of ion currents in the voltage clamp mode.

Publication link
2011 – Automated Patch Clamp on mESC-Derived Cardiomyocytes for Cardiotoxicity Prediction
Patchliner and Port-a-Patch Publication in Journal of Biological Chemistry (2011) Authors: Stoelzle S., Haythornthwaite A., Kettenhofen R., Kolossov E., Bohlen H., George M., Brüggemann A., Fertig N.

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro–generated stem cell–derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell–derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell–derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

Publication link
2008 – Ion channel screening – automated patch clamp on the rise
Port-a-Patch and Patchliner Publication in Drug Discovery Today (2008)   Authors: Farre C., George M., Brüggemann A., Fertig N.

Ion channel proteins are of major importance for the human physiology and thus highly attractive molecular drug targets. Large-scale ion channel screening of wanted and unwanted drug effects is required, but has been limited by the lack of adequate screening technology, because available methods put a tradeoff between high-throughput and high-information content. The advent of automated patch clamp platforms has revolutionized ion channel screening, enabling investigations from a more functional perspective at a much higher throughput. The current status of automated patch clamp platforms, their strengths and drawbacks as well as future developments are reviewed.

Publication link
2009 – Port-a-Patch and Patchliner: High fidelity electrophysiology for secondary screening and safety pharmacology
Port-a-Patch and Patchliner Publication in Combinatorial Chemistry & High Throughput Screening (2009) Authors: Farre C., Haythornthwaite A., Haarmann C., Stoelzle S., Kreir M., George M., Brüggemann A., Fertig N.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately four years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world’s smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion’s higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and NaV1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Publication link
2007 – Automated ion channel screening: patch clamping made easy
Port-a-Patch and Patchliner Publication in Expert Opinion Therapeutic Targets (2007) Authors: Farre C., Stoelzle S., Haarman C., George M., Brueggemann A., Fertig N.

Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion’s electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.

Publication link
2007 – Planar Patch Clamping
Port-a-Patch and Patchliner book chapter in "Patch Clamp Analysis – Advanced Techniques", Series: Neuromethods (2007) Authors: Behrends, J.C., Fertig, N.

The technique of patch clamping can be seen in retrospect as a combination of two separate lines of development that both originated in the 1960s and 1970s. The classical biophysics of the nerve impulse had by then been established in the squid giant axon using a combination of (1) voltage clamping with axial wire electrodes and (2) internal perfusion or dialysis. This combination had given experimenters control of both the electrical and the chemical gradients governing membrane ion flux. The problem of the day was to extend this type of analysis to smaller, noncylindrical, cellular structures (such as neuronal somata) that would not allow insertion of metal wires, let alone tolerate any of the procedures used for internal perfusion or dialysis of squid axons. While intracellular glass microelectrodes afforded intracellular electrical access to most cellular somata, two independent electrodes for current passing and voltage recording, respectively, were initially necessary, until time-sharing systems made single-microelectrode voltage clamping possible. Even then, however, two severe problems remained: (1) spatially nonuniform voltage control (the so-called space-clamp problem), and (2) the lack of control over intracellular ionic composition.

Publication link
2006 – Microchip technology for automated and parallel patch clamp recording
Port-a-Patch and Patchliner Publication in Small Journal (2006) Authors: Brüggemann A., Stoelzle S., George M., Behrends J.C., Fertig N.

The patch-clamp technique is the state-of-the-art technology for the study of a large class of membrane proteins called ion channels. Ion channels mediate electrical current flow, have crucial roles in cellular physiology, and are important drug targets. However, patch clamping is a laborious process requiring a skilled experimenter and is, therefore, not compatible with the high throughput needed in drug development. The solution for automated and parallel patch-clamp measurements that is provided by microchip technology is presented here.

Webinar
2017 – The CiPA initiative – Origin, Idea, Status, Issues, and Impact on Drug Development
Presenter:
Dr. Herbert Himmel, Bayer Wuppertal, Germany

Source:
Nanion User Meeting
October 25.-26. 2017
Webinar
14.10.2020 | Webinar: Pharmacological Characterization of Natural Tobacco Alkaloids in the Presence of Positive Allosteric Modulators Against Humana4b2 and a7 Nicotinic Acetylcholine Receptors
Dr. Omar Alijevic (Philip Morris International)


This is an on-demand webinar from Nan]i[on and Friends 2020.

The pharmacological profile of tobacco alkaloids is essential for understanding their physiological effects. Nicotinic acetylcholine receptors (nAChRs) are an important target of tobacco alkaloids with primarily agonistic effects reported for α4β2 nAChRs, but with minimal evidence of α7 activity. In this study, we used a membrane potential assay and automated patch-clamp electrophysiological approaches to functionally characterize distinct groups of tobacco alkaloids in the presence of a subunit-specific positive allosteric modulator (PAM) of human α4β2 and α7 nAChRs. We screened a total of 71 tobacco alkaloids, of which 16 were active against α4β2 and 11 against α7 nAChRs.

The most abundant alkaloids in tobacco leaves—namely nicotine, nornicotine, anabasine, (S)-anatabine, and (R)-anatabine—exhibited potencies (EC50alkaloid+PAM) of 0.02-20 μM against α4β2 and 0.2-10 μM against α7 nAChRs. In the presence of the PAM, nicotine and anabasine, respectively, were found to be the most potent α4β2 and α7 nAChRs agonists. Relative to (S)-anatabine, (R)-anatabine was 5-fold more potent against α4β2; the relationship was found to be inverse in case of α7 nAChRs. In addition, 13 alkaloids demonstrated agonistic effects only in the presence of the PAM and were, therefore, considered to be silent agonists. In conclusion, the data revealed 17 naturally occurring tobacco alkaloids that exhibited a dramatic increase in potency against human α4β2 and α7 nAChRs in the presence of PAMs (relative to that in the absence of the PAM). Our study recognized a subunit-specific enantiomer preference of anatabine and identified several alkaloids with silent agonist properties for human α4β2 and α7 nAChRs.

Webinar
16.10.2020 | Webinar: Voltage-Gated Ion Channels Fenestrations as a Drug Access Pathway
Prof. Tamer M. Gamal El-Din (University of Washington)


This is an on-demand webinar from Nan]i[on and Friends 2020.

Voltage-gated sodium channels initiate electrical signals in nerve and cardiac muscle, where their hyperactivity causes pain and cardiac arrhythmia. Local anesthetics and antiarrhythmic drugs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. We studied an ancestral bacterial sodium channel to elucidate the structure of the drug-binding site and the pathway for drug entry to the receptor site. We found that the drug-binding site is located in the center of the transmembrane pore, through which sodium ions move and fenestrations form an access pathway for drug entry directly from the cell membrane. These results show how these widely used drugs block the sodium channel and have important implications for structure-based design of next-generation drugs. In my talk, I'll also shed light on the fenestrations of other ion channels to argue that fenestrations could be an entry pathway for many ion channels.

Webinar
11.05.2013 | Webinar: Patchliner – unlimited experimental freedom
This Webinar covers the features of the Patchlliner, including the "minimized cell usage" for expensive cells.

The presentation will address the general setup and special features of the Patchliner from Nanion. One highlight is the recently published procedure to minimize cell usage for stem cell-derived and primary cells on our most flexible automated patch clamp device. Furthermore, fast perfusion, temperature control capabilities and current clamp are discussed and case studies are presented.

Webinar
14.07.2022 | Webinar: An in silico-in vitro pipeline for drug cardiotoxicity screening identifies ionic proarrhythmia mechanisms
Presenter:
Dr. András Horváth, Application Scientist (Nanion Technologies)

Title:
Automating action potential pharmacology in hiPSC cardiomyocytes

Presenter:
Alex Clark, Ph.D. Student - Christini Lab (Cornell University)

Title:
An in silico-in vitro pipeline for drug cardiotoxicity screening identifies ionic proarrhythmia mechanisms

All new drugs are screened for their proarrhythmic potential using a method that is overly conservative and provides limited mechanistic insight, which can lead to the misclassification of beneficial drugs as proarrhythmic. Here, we developed an in silico-in vitro pipeline to circumvent these shortcomings. An iPSC-CM computational model was used to design electrophysiological voltage-clamp (VC) protocols for use during in vitro drug studies. Such VC data, along with AP recordings, were acquired from iPSC-CMs before and after treatment with a control solution or verapamil, cisapride, quinine, or quinidine. AP prolongation was seen in response to quinidine and quinine. The VC protocol identified all strong IKr blockers. The protocol also detected block of ICaL by verapamil and Ito by quinidine. The VC data also uncovered a previously unidentified If block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp.

Webinar
28.04.2020 | Webinar: Validation and optimization of automated patch clamp voltage-gated Ca2+ channel assays
Speakers:
Dr. Marc Rogers (Chief Scientific Officer, Metrion Biosciences)
Dr. András Horváth (Application Scientist, Nanion Technologies)

Marc will outline the development, optimization and validation of a range of voltage-gated Ca2+ channel assays on the Patchliner automated patch clamp platform that were subsequently used in an 8 year drug discovery collaboration between Metrion Biosciences and a german pharma company.

The project was successful in identifying several lead series of selective,state-dependent inhibitors of Cav2.2 N-type channels for use as novel, non-opioid analgesic.This required the creation of biophysical screening assays to identify the potency of small molecules against the resting and inactivated state of the human neuronal Ca2+ channels Cav2.1(P/Q type), Cav2.2 and Cav3.2 (T-type), some of which are notoriously difficult in terms of expression levels and current rundown.

Webinar
27.06.2017 | Webinar: New Dynamics in Automated Patch Clamp
This webinar shows new applications on dynamic patch clamp of iPSC-derived cardiomyocytes and introduces an assay on KCa3.1 expressed in erythrocytes.

Speakers:

Dr. Teun P. de Boer, University Medical Center Utrecht

Maria Giustina Rotordam, Nanion Technologies

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are an interesting source of cells for safety pharmacology, but there are caveats that have to be taken into account. A typical property of hPSC-CM is automaticity, the cardiomyocytes beat spontaneously, resembling cells in the sinoatrial node of the heart. The spontaneous beating is due to lack of IK1 ion channels, which normally assure a stable resting membrane potential in cardiomyocytes. Work by our group has demonstrated that the depolarized state of hPSC-CM limits their usefulness in assays aimed at detecting proarrhythmic properties of drugs (Jonsson et al., 2012, PMID: 22353256).
Dynamic clamping can provide virtual, simulated IK1 channels to a real biological hPSC-CM in a patch clamping experiment. Key benefits of this approach include full control of the added IK1 conductance to each cardiomyocyte, it can be applied to any hPSC-CM source and it can be coupled to automated patch clamping machines. In this webinar I will discuss our work on using the dynamic clamping technique with a Patchliner.

Tutorial video
2018 – Igor Tutorial for Patchliner Data Analysis
Presenter:
Dr. Nadine Becker, Product Manager Patchliner, Nanion Technologies GmbH, Germany

This Igor video tutorial for Patchliner data analysis demonstrates how concentration-response curves are quickly displayed and analyzed. We show how data are loaded and displayed, and the normalized data are fitted and averaged to obtain IC50 values. Please note that more details and features are described in our QuickStart Guide for Igor.

User meeting video
15.10.2020 | Webinar: Evaluation of possible proarrhythmic potency: variability of IC50 values of drugs under different conditions and in different platforms
Dr. Péter Orvos (University of Szeged), This is an on-demand webinar from Nan]i[on and Friends 2020.

The conventional microelectrode technique and the manual patch clamp method offer direct, information-rich, and real-time in vitro technologies to study proarrhythmic effect of drugs and drug candidate compounds. Although providing excellent data quality, these tests are complicated, time consuming and expensive for the large numbers of compounds. Automated patch-clamp platforms are mainly used with stably expressing cell lines and suitable for rapid and high-quality pharmacological investigation of drug candidates. The Comprehensive in Vitro Proarrhythmia Assay (CiPA) was initiated to further improve these preclinical drug safety paradigms. However, some evidence indicates that the different proarrhythmic pharmacological assays result in contradictory outcomes raising serious questions regarding their predictability for in vivo situations including clinical settings. IC50 values may varied between platforms, therefore, aim of our study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch clamp methods on HEK293 cells. The native ion current (IKr) were recorded from rabbit ventricular myocytes by manual patch clamp technique.

Dofetilide, cisapride, sotalol, terfenadine and verapamil were tested in hERG assay at both room temperature and 37°C with Patchliner. All these compounds were more potent at physiological temperature and therefore, it is a desirable option to study hERG currents at physiological temperature. To evaluate the prognostic value of hERG assay these agents were subjected for further investigations. The IKr current blocking capability of the compounds was tested on rabbit ventricular myocytes with manual patch clamp method at 37°C. The corresponding IC50 values of dofetilide, cisapride and verapamil were in good agreement with IC50 values obtained with Patchliner in hERG assays. As sotalol and terfenadine have stronger effect on IKr measured by manual patch clamp method compared with hERG automated patch clamp experiments, the effects of these drugs on hERG current using manual patch clamp technique were also investigated to study how the potency of these drugs are influenced by the experimental techniques themselves. In contrast with the hERG automated patch clamp assays, the effects of sotalol and terfenadine on hERG current were stronger measured by the manual patch-clamp technique.

In conclusion, results obtained with automated patch-clamp equipment in HEK-hERG cells usually show a reasonable conformity with outcomes of IKr current experiments. The Patchliner system used in our study is well suited to perform safety pharmacological studies. Variability of IC50 values of drugs in different platforms observed in certain cases, which could have been caused by the lack of continuous flow of compound-containing solutions.

Product video
Patchliner- 2021 (Product video demo)
The Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously.

The Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. With its vast experimental freedom and gigaseal data quality, the Patchliner is one of the most versatile patch clamp instruments on the market.

Product video
Patchliner- 2022 (Product video demo)
The Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously.

The Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. With its vast experimental freedom and gigaseal data quality, the Patchliner is one of the most versatile patch clamp instruments on the market. The Patchliner is versatile yet robust, ideal for basic research of ion channel biophysics and mechanisms of action, and sophisticated assays including heat activation of TRP channels, activation of Ca2+-activated channels by internal exchange and fast external solution exchange with minimal exposure for ligand-gated ion channels such as nAChα7. The Patchliner is also an excellent tool for routine assays such as safety screening of hERG or other cardiac ion channels in line with the CiPA initiative. Since its introduction in 2006, over 100 instruments have been installed worldwide, in academic labs (46%), pharmaceutical companies (34%) and CROs (20%). The Patchliner is appreciated because of routine high success rates (greater than 80% gigaseals), optimized assays including minimized cell consumption and proven use of primary cells and stem cells. Our dedicated team of electrophysiologists and engineers are committed to continuous in-house assay development, software and hardware advancements, ensuring fast and custom-tailored solutions for your assay demands.

Poster PDF
2022 – Using single-chain variable fragments (scFv) to map the β3-subunit binding site on the pain-sensing sodium channel Nav1.7
Patchliner Poster, Cambridge Ion Channel Forum 2022
Product Sheet PDF
Patchliner – Product Sheet
Flyer PDF
Patchliner Product Flyer – Safety Edition
Poster PDF
2020 – Automated Patch Clamp System Introducing Simulated Ik1 Into stem cell derived Cardiomycoytes Using Dynamic Clamp
Patchliner poster, 64th Annual Meeting of the Biophysical Society
Flyer PDF
Patchliner Product Flyer – Dynamite8
Flyer PDF
Patchliner Product Flyer – PatchControl HT Release 2021
Application Note PDF
TRPV3 – “Heat activation of TRPV3 on Nanion’s Patchliner”
Patchliner application note:   

The transient receptor potential cation channel, subfamily V, member 3 (TRPV3), is a ligand-gated, nonselective cation channel first described in 2002. It exhibits 43% sequence identity to TRPV1. Although TRPV3 has been detected immunologically in the CNS and suggested to be often co-localized with TRPV1, it is found more robustly in keratinocytes in skin and, given it’s threshold for temperature activation of >34˚C, it has been speculated that TRPV3 may act in co-operation with sensory afferents to perceive warmth and signal elevated temperature. TRPV3 can also be activated by the ligand 2-Aminoethoxydiphenyl borate (2-APB). The TRPV3 channel, along with other TRPV channels, may play an important role in chronic pain and, therefore, is receiving increasing attention as a potential therapeutic candidate for the treatment of chronic pain. Here we present data collected on a 4-channel Patchliner with temperature control showing the potential use of the Patchliner to record TRPV3 currents activated by 2-APB or heat. As previously reported, TRPV3 currents sensitize to repeated applications of 2-APB or heat, a phenomenon we also observed. At low concentrations of 2-APB, the currents were primarily outwardly rectifying but at higher concentrations and with prolonged exposure they often became dual rectifying (data not shown). This is also in good agreement with the literature. In contrast, the temperature-activated responses were always outwardly rectifying with little inward current. The inward currents activated by 2-APB could be blocked by ruthenium red (RR) as expected.

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TRPM8 – “TRPM8 activation by menthol and Eucalyptol performed on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Charles River.

TRPM8 is a member of the transient receptor potential channel (TRP) family. TRPM8 is known to be a thermosensitive channel, activated by cold temperatures (below ~25˚C) and ligands such as menthol, Eucalyptol and icilin. It belongs to the melastatin subfamily of TRP channels and shows an outward rectification with a relatively high permeability for calcium ions and little selectivity between monovalent cations. Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, where the TRPM8 channel is specifically expressed. Here we present data of hTRPM8 collected on the Patchliner. Cells performed well on the Patchliner with a success rate of 89% for seal resistance >600 MΩ. Channel activation by the agonists menthol and Eucalyptol is shown.

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TRPV1 – “Heat activation of TRPV1 on Nanion’s Patchliner”
Patchliner application note:   

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1), is a ligand-gated, non-selective cation channel widely expressed in the peripheral and central nervous system. The TRPV1 channel can be activated by a number of physical and chemical stimuli, including capsaicin (the active ingredient in chili peppers), noxious heat (typically >42˚C) and low pH. The TRPV1 channel is putatively involved in the perception and transmission of painful stimuli. Importantly, this channel is proposed to underlie many chronic pain states including inflammation, neuropathic pain and cancer pain, amongst others. These types of pain states are currently poorly managed by the pain medications available and this has led the pharmaceutical industry to seek novel targets for pain management, such as TRPV1. However, TRPV1 antagonists have so far failed in clinical trials due to an undesirable increase in core body temperature resulting in hyperthermia. From these studies, it is proposed that tonically active TRPV1 channels are involved in maintaining normal body temperature and this could have significant influences on drug design. Finding novel compounds with differing effects on capsaicin activation and heat activation may be crucial in the discovery of lead compounds for the treatment of pain and other disease states. Here we present data collected on the 4-channel Patchliner with temperature control showing the potential use of the Patchliner® to record capsaicin and heat activation of TRPV1 channels and subsequent block by ruthenium red (RR).

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P2X3 – “Activation and inhibition of P2X3 channels recorded on the Patchliner”
Patchliner application note:   Cells were engineered and kindly provided by Axxam S.p.A., Milan.  

P2X receptors are ligand-gated ion channels that open in response to extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. P2X receptors are found on many cell types including smooth muscle cells, sensory neurones, epithelia, bone and leukocytes. A role for P2X receptors has been suggested in transmission of thermal stimuli, chemosensory signalling, taste and pain. To date, 7 P2X receptor genes have been cloned and studied in heterologous expression systems. Functional receptors are trimeric, which can be homomeric or heteromeric. The P2X2 and P2X3 receptors can function either as homomers or as P2X2/3 heteromers. When expressed together, a mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to exist, which may be distinguished through their biophysical and pharmacological properties. Both P2X3 homomers and P2X2/3 heteromeric receptors have been implicated in nociception and pain signalling and may be important therapeutic targets for analgesic drugs. The P2X3 and P2X2/3 receptor antagonist MK-7264 (gefapixant), has recently progressed to Phase III trials for refractory or unexplained chronic cough.Here we present data collected on the Patchliner showing activation and inhibition of P2X3 currents expressed in CHO cells with rapid and brief application of ligand (using the stacked solution approach). αβ-methylene ATP (αβ-MeATP) activated P2X3 receptors in a concentrationdependent manner with an EC50 value similar to those in the literature. P2X3 receptors could be repetitively activated by αβ-MeATP and blocked by A-317491 with an IC50 value in good agreement with the literature.

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Neurons – “Stem cell-derived neurons recorded on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Cellular Dynamics.

Cellular Dynamics International is developing iCell® Neurons, human iPS cell-derived neurons. These neurons have been used on Nanion’s Patchliner, an automated patch clamp device for recording from up to 8 cells simultaneously. Cells were thawed and cultured as per the manufacturer’s instructions. After plating, cells developed neuronal outgrowth within 24 hours and could be kept in culture for 2 weeks. An investigation into voltage- and ligand-gated ion channels expressed in these cells was undertaken using the Patchliner. The first experiments are shown here and offer promising results for combining a cellular model for neurons and an increased throughput patch clamp device.

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P2X2 / P2X3 – “P2X2/3 receptors recorded on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Evotec.  

P2X receptors are ligand-gated ion channels that open in response to extracellular ATP. They are permeable to small monovalent cations, some having significant divalent or anion permeability. P2X receptors are found on many cell types including smooth muscle cells, sensory neurones, epithelia, bone and leukocytes. A role for P2X receptors has been suggested in transmission of thermal stimuli, chemosensory signalling, taste and pain. To date, 7 P2X receptor genes have been cloned and studied in heterologous expression systems. Functional receptors are trimeric, which can be homomeric or heteromeric. The P2X2 and P2X3 receptors can function either as homomers or as P2X2/3 heteromers. When expressed together, a mixture of P2X2 and P2X3 homomers as well as P2X2/3 heteromers are likely to exist, which may be distinguished through their biophysical and pharmacological properties. P2X2/3 receptors have been implicated in nociception and pain signalling and may be important therapeutic targets for analgesic drugs.Here we present data collected on a 4- or 8-channel Patchliner showing the potential use of the Patchliner to record P2X2/3 currents activated by ATP. ATP activated P2X2/3 receptors in a concentration-dependent manner with an EC50 similar to those reported in the literature for a mixture of homomeric and heteromeric P2X2/3 receptors. The currents desensitized slowly confirming that they are mediated by P2X2 and P2X2/3 receptors rather than P2X3 receptors. P2X2/3 receptors could be repetitively activated by ATP and blocked by suramin with an IC50 in good agreement with the literature.

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NaV1.8 – “Characterization of rNaV1.8 (ND7-23) on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Millipore.

The NaV1.8 gene (originally named PN3 or SNS; gene symbol SCN10A) encodes a voltage-gated sodium (NaV) channel, selectively expressed in dorsal root ganglion (DRG) neurons. DRGs transmit peripheral stimuli to the central nervous system and are involved in nociception. Different NaV channels play a key role in modulation of DRG action potentials. In particular, the fast upstroke of the action potential is mediated by NaV channels. NaV channels are in part characterized by their TTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]). NaV1.8 is a TTXr channel. Compared with other NaV channels, NaV1.8 has slow activation and inactivation kinetics and is opened at relatively high  voltages. It is an interesting drug target for inflammatory and neuropathic pain, because modulation of NaV1.8 by inflammatory mediators seems to be a key mechanism of DRG nociceptor sensitization and activation. Interestingly, NaV1.8 has been reported to play an important role in the perception of cold pain. In this Application Note we present data recorded on the Patchliner characterizing ND7-23 cells (a rat DRG/mouse neuroblastoma hybrid) stably transfected with rat NaV1.8. All experiments were performed in the presence of 100 nM TTX to block the endogenous TTXs Na+ current present in these cells. The NaV1.8 activation and inactivation properties and tetracaine sensitivity recorded on the Patchliner were consistent with those reported in the literature.

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Neurons – “Peri.4U and Dopa.4U stem cell-derived neurons recorded on Nanion´s Patchliner”
Patchliner application note:   Cells were kindly provided by Axiogenesis.  

Human induced pluripotent stem cell-derived neurons (hiPSC-neurons) may provide a viable cellular model for studying the mechanisms underlying neurological diseases and drug development. Axiogenesis provides a number of hiPSC-neurons including dopaminergic neurons (Dopa.4U) and peripheral neurons (Peri.4U), amongst others. These neurons have been used on in-vitro systems such as multielectrode arrays (MEA), immunocytochemistry and calcium imaging. They are an interesting model for studying neurological diseases such as Parkinson’s Disease, as well as for efficacy, drug discovery and toxicity studies. In this study, the Patchliner was used to record from Dopa.4U and Peri.4U neurons in voltage and current clamp modes. The cell harvesting procedure was optimized (using papain) to ensure that the cells retained proximal dendrites and initial axon segments in order to maintain ion channel expression present in these regions. Due to their irregular shape (presence of processes), success rate (typically 20 - 50% for RSeal >200 MΩ) was lower than other cell types which have a smooth, round shape, e.g. standard cell lines. Voltage-gated Na+ (NaV) and K+ (KV) currents were recorded in both cell types. Action potentials (AP) were also recorded and block of the AP of Dopa.4U cells by lidocaine is shown.

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KV1.3 – “Pharmacological analysis of heterologous and endogenous expressed KV1.3 channels in Sf21 insect cells and T-lymphocytes”
Patchliner application note:   Cells were kindly provided by conoGenetix.

The voltage-gated potassium channel KV1.3 is expressed in human naive, central memory T cells (TCM), and effector memory T cells (TEM). TEM cells are important mediators of a variety of chronic inflammatory autoimmune diseases including multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and vasculitis. Antigenic stimulation of TEM cells results in upregulated KV1.3 channel expression. It is proposed that a selective block of KV1.3 channels leads to an inhibition of functionality of TEM cells without affecting the function of naive and TCM cells. Thus, KV1.3 is a promising therapeutic target that could be used to efficiently medicate chronic autoimmune diseases without generating typical side effects associated with today’s immunosuppressive therapies. In the laboratory of conoGenetix biosciences the insect cell line Spodoptera frugiperda (Sf21) is used in combination with a Baculovirus infection system for the heterologous expression of recombinant ion channels. Sf21 cells offer highly specific protein expression and correct posttranslational modifications resulting in functional channels with comparable electrophysiological parameters of ion channels in vivo. In this Application Note we compare the electrophysiological properties of the potassium selective ion channel KV1.3 expressed in Sf21 cells and endogenously expressed KV1.3 channels in TEM cells. For the pharmacological analysis the 4 - channel Nanion Patchliner and one of our innovative, KV1.3 specific, proprietary peptides was used.

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NaV1.7 – “Pharmacology on hNaV1.7 performed on Nanion’s Patchliner at Vhalf “
Patchliner application note:   Cells were kindly provided by Anaxon.

The NaV1.7 gene (SCN9A) encodes a voltage-gated sodium (NaV) channel, primarily expressed in the peripheral nervous system and has been isolated from rat dorsal root ganglion (DRG) neurons, human medullary thyroid cancer cells (hNE-Na) and PC12 cells. Different NaV channels play a key role in modulation of action potentials in the central and peripheral nervous systems. In particular, the fast upstroke of the action potential is mediated by NaV channels.NaV channels are in part characterized by their TTX-sensitivity (TTX-resistant [TTXr], TTX-sensitive [TTXs]). NaV1.7 is a TTXs channel and is sensitive to TTX in the nanomolar range. The role of hNaV1.7 has yet to be fully elucidated but is proposed to play an important role in nociception and pain sensing. NaV1.7 has been implicated to play a role in disease pain states, in particular inflammatory pain and hypersensitivity to heat following burn injury. Common to many of the voltage-gated ion channels, a number of compounds display a higher affinity for the inactivated state of the channel. For this reason, it is important to be able to reliably measure the effects of compounds at Vhalf of  inactivation, the voltage at which 50% of the channels are inactivated. In this Application Note we present data using an 8-channel Patchliner characterizing CHO cells stably expressing hNaV1.7. The hNaV1.7 activation and inactivation properties are consistent with those reported in the literature. The potency of sodium channel blockers mexiletine, tetracaine, amitriptyline and lidocaine were compared using a holding potential of -120 mV vs the Vhalf of inactivation.

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hERG – “Effect of temperature on erythromycin action on hERG currents recorded on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Millipore.

The hERG gene encodes a potassium channel responsible for the repolarization of the IKr current in cardiac cells (Sanguinetti et al, 1995). This channel is important in the repolarization of the cardiac action potential. Abnormalities in this channel can lead to long or short QT syndrome, leading to potentially fatal cardiac arrhythmia. Given the importance of this channel in maintaining cardiac function, and disturbances of channel activity by certain compounds such as anti-arrhythmias and anti-psychotics, it has become an important target in compound safety screening. Compounds can display different properties or different potencies at physiological temperature (35°C) vs. room temperature (RT) and therefore, it is a desirable option to be able to study this channel electrophysiologically at elevated temperature. One such compound which has been shown to have an increase in potency at physiological temperature is erythromycin. Erythromycin is a macrolide antibiotic which can cause QT prolongation and cardiac arrhythmia. Erythromycin has been shown to block hERG channels at physiological temperature with an IC50 of approximately 40 mM (Stanat et al, 2003; Duncan et al, 2005). However, at RT erythromycin is much less potent. At a concentration of 100 mM, erythromycin causes no significant block of hERG currents at RT but significantly blocks currents at physiological temperature (Guo et al, 2005). Here we present data collected on an 8-channel Patchliner with temperature control at RT and at 35°C and the effect this has on the potency of erythromycin.

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KCa3.1 – “Modulation of hKCa3.1 by internal Ca2+ performed on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Charles River.

The intermediate-conductance calcium-activated K+ channel, also known as KCa3.1, IKCa1 or SK4, is a member of the large family of potassium channels gated by calcium. It can be distinguished from the other calcium-activated K+ channels by differences in channel conductance, calcium sensitivity, voltage dependence and pharmacological properties. The hKCa3.1 channel is encoded by the KCNN4 gene. It is primarily expressed in peripheral tissues, including those of the hematopoietic system, colon, lung, placenta, and pancreas and has been proposed to play an important role in a variety of physiological processes including volume regulation in erythrocytes, proliferation and differentiation of B- and T-lymphocytes, and tissue protection following spinal cord injury. Importantly, the hKCa3.1 channel is a promising therapeutic target for a variety of health disorders including sickle cell anaemia and immunological disorders. The Ca2+-binding protein, calmodulin (CaM), is required for the activation of hKCa3.1. The Ca2+-CaM complex is proposed to bind to an intracellular domain of the C terminus of all subunits in the tetramer, inducing conformational changes to open the channel. In this study the Patchliner was used to perform a biophysical and pharmacological characterization of hKCa3.1 channels expressed in CHO cells. hKCa3.1 was activated by exchanging the internal solution to a solution containing free-Ca2+ and blocked by external application of non-selective (BaCl2) and selective (TRAM-34) inhibitors with an IC50 value consistent with that reported in the literature.

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GABAA (a1b2g2) – “Rapid agonist applications in precise intervals (GABAA) “
Patchliner application note:   

HEK-cells expressing GABAA-receptors were investigated with the Patchliner using a stacked application approach for rapid administration of compounds to the patch-clamped cells. The GABAA receptor family is the most important class of inhibitory ion channels involved in synaptic transmission, and are selectively permeable to monovalent anions. They constitute an important therapeutic area for drugs affecting anxiety, sleep and muscle relaxation. As with most ligand gated ion channels, GABAA exhibit receptor desensitization, which is a common phenomenon for ligand gated ion channels. Desensitization can be either exposure time dependent or concentration dependent, or both. Desensitization and recovery kinetics varies from milliseconds to tens of minutes, all depending on receptor type and subunit composition. For rapidly desensitizing ion channels, it is important that compound application is fast, so that the entire ion channel population is exposed to maximum concentration before entering the desensitized state. Exposure time and application intervals are important factors affecting desensitization and recovery from desensitization, to minimize deletrious effects or receptor desensitization. In the experiments presented here, compounds were added to the cells with accurate timing. In addition, a method for brief compound application was developed, allowing 1 s pulses of compound to the patch clamped cells. In this way, receptor desensitization can be minimized, and because of the timed exposures, effects of desensitization.

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Glycine receptors (GlyRa1) – “Modulators for glycine receptors investigated using the Patchliner “
Patchliner application note:   Cells were kindly provided by AstraZeneca.

The Patchliner was used to study the pharmacology of the hGlyR a1 receptor expressed in mouse fibroblast cell line (L-tk). The Patchliner allows for fast application of drugs (50 ms) with precisely controlled application intervals and wash times. These are important experimental parameters when investigating ligand gated ion channels and their effectors since many ligand gated ion channels rapidly desensitize. Kinetics and level of desensitization are determined by ligand concentration, exposure time, or both. Because of the Patchliner's rapid solution exchange combined with brief drug exposure, capabilities and timed application intervals, the deleterious effect caused by receptor desensitization can be minimized and corrected for. Here we show the use of Nanion's Patchliner for long (22 s) and short (1 s) application of glycine to patch clamped whole-cells. For the short applications of compound, a stacked solution application protocol was used. Specifically, two zones of solution were aspirated into the pipette used for administration. When applied to the cell, this results in exposure first to the agonist zone followed by a rapid wash out after 1 s of drug application. In all the protocols used, pre-incubation of modulators is possible, as well as washout in the presence of a modulator.

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CFTR – “Effect of internal F- on activation of Cystic Fibrosis Transmembrane Conductance (CFTR) regulator by forskolin ” (report)
Patchliner application report:   

The aim of this study was to investigate the effect of internal F- on the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) by forskolin in the whole cell patch clamp configuration. Experiments were conducted with Nanion’s fully automated patch clamp device, the Patchliner.

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GABAA (a1b2g2) – “Patchliner – Investigating the pharmacology of GABAA receptors expressed in HEK293 cells”
Patchliner application note:   

γ-amino butyric acid (GABA), β-alanine and bicuculline were investigated on GABAA-receptors expressed in HEK293 cells. The GABAA receptor family is the most important class of inhibitory ion channels involved in synaptic transmission. GABAA receptors are selectively permeable to monovalent anions. They constitute an important therapeutic target for drugs affecting anxiety, sleep and muscle relaxation. In this study, the cells were exposed to compound for 30 s, followed by a 60 s wash step. Solution exchange around the cell was fast, in the order of 50 ms for saturating concentrations. Complete and rapid switching is important when investigating ligand gated ion channels, since the response is often very fast and most receptors desensitize. For rapidly desensitizing ion channels, fast compound application is crucial, so that the entire ion channel population is exposed to maximum concentration before entering the desensitized state. Desensitization and recovery kinetics vary from milliseconds to tens of minutes, depending on receptor type and subunit composition as well as exposure time and/ or compound concentration. Recordings were made in the whole-cell configuration, using the Patchliner (4 amplifier channels). Expected pharmacology was obtained for the investigated compounds.

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CaV2.2 – “Characterization of CaV2.2 (HEK293) on Nanion’s Patchliner”
Patchliner application note:   

The voltage gated N-type calcium channel (CaV2.2) is encoded by the gene CACNA1B. CaV2.2 is a high voltage activated calcium channel. CaV2.2 is found mainly in the brain, where it mediates neurotransmitter release at the synapse. The strong depolarization of neuronal action potentials causes the opening of the channel. Calcium can then enter the cell and initiates the fusion of the neurotransmitter vesicles with the membrane. CaV2.2 is inhibited by w-conotoxin, a neurotoxin of the fish hunting snail, with high specificity. CaV2.2 has been implicated in the transmission of pain. Pharmacological block of CaV2.2 by compounds based on w-conotoxin has been shown to be effective against strong chronical pain. The biophysical and pharmacological properties of the cells are presented in this Application Note.

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CaV3.2 – “Characterization of CaV3.2 (HEK293) on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Millipore.

The gene CACNA1H encodes the α1H subunit of the voltage-gated calcium channel CaV3.2. It belongs to the low voltage-activated T-type calcium channels. CaV3.2 displays the typical characteristics of the T-type channels: activation at low depolarization of the membrane and transient kinetics. T-type Ca2+ channels are involved in diverse, mainly rhythmic processes like e.g. pacemaking and generation of thalamocortical rhythms in sleep or epilepsy. CaV3.2 is expressed in a wide variety of cells. Amongst others it has been found in kidney, smooth muscle, brain, adrenal and cardiac cells. It seems to be involved in contraction of smooth muscle and the secretion of the adrenal hormones aldosterone and cortisol. Pharmacological block of T-type channels may lead to new drugs for the treatment of hypertension and epilepsy. The biophysical and pharmacological properties of the cells are presented in this Application Note.

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Cardiomyocytes – “Voltage and current clamp recordings of Cellartis hiPS-CM on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Takara Bio Europe AB.  

Human induced pluripotent stem (iPS) cell-derived cardiomyocytes have the potential to provide the ultimate model system for identifying potential anti-arrythmic effects of drugs during routine safety screening. Takara Bio Europe AB is providing a human iPS cell-derived cardiomyocyte product line for use in testing the efficacy and safety of pharmaceutical therapies. The ability to characterize the ion channel profile of these cells and record action potentials at a reasonable throughput is essential to fully realise the potential of this kind of product line. Building on the success of recording stem cells on the Patchliner, Cellartis® hiPS-CM, human iPS cell-derived cardiomyocytes, have now been characterized on the Patchliner in the voltage and current clamp mode. In this Application Note we present data using an 8-channel Patchliner. In the voltage clamp mode, voltage-dependent Na+ (NaV), K+ (KV) and Ca2+ (CaV) channel currents were recorded. As expected, action potentials could be elicited in the current clamp mode. Furthermore, spontaneous action potentials could be recorded as well.

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Cardiomyocytes – “Voltage and current clamp recordings of Cor.4U human iPS cell-derived cardiomyocytes on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Axiogenesis.

Although mouse embryonic stem (ES) cell-derived cardiomyocytes, e.g. Cor.At® cells from Axiogenesis, can provide a useful model for drug discovery and safety testing as an alternative to acutely dissociated rat or mouse cardiomyocytes, human induced pluripotent (iPS) cell-derived cardiomyocytes have the potential to provide the ultimate model system for identifying potential antiarrythmic effects of drugs during routine safety screening. Axiogenesis has now launched the Cor.4U® human iPS cell-derived cardiomyocyte product line for use in testing the efficacy and safety of pharmaceutical therapies. The ability to characterize the ion channel profile of these cells and reliably record action potentials at a reasonable throughput is essential to fully realise the potential of this kind of product line. Building on the success of Cor. At® mouse embryonic stem (ES) cell-derived cardiomyocytes on the Patchliner, Cor.4U® human iPS cell-derived cardiomyocytes have now been characterized on the Patchliner in the voltage and current clamp modes. In this Application Note we present data using an 8-channel Patchliner characterizing Cor.4U® cells. In the voltage clamp mode, voltage-dependent Na+ (NaV), K+ (KV) and hERG (an inward current using a high K+-containing external solution) channel currents were recorded (Fig. 1). When the Ca2+ channel agonist BayK 8644 was used a voltage-gated Ca2+ (CaV) current could be recorded. As expected, action potentials could be elicited in the current clamp mode. The effect of the compounds TTX and BayK 8644 on the action potentials evoked in Cor.4U cells is also shown.

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ASIC3 – “Characterization of hASIC3 (HEK) on Nanion´s Patchliner”
Patchliner application note:   Cells were kindly provided by Millipore.

Acid-sensing ion channels (ASICs) are ligand-gated ion channels activated by protons and are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family. They are highly sensitive to extracellular acidosis. In rodents, where they are mainly expressed in neurons of the peripheral nervous system, ASIC3 plays an important role as sensor of non-adaptive pain which is correlated to tissue acidosis. However, the role of the human ASIC3 channel has not yet been elucidated. In contrast to other ASIC ion channels, ASIC3 shows a sustained window current upon external acidification. Here we present data recorded on an 8-channel Patchliner. Current responses upon external acidification as well as amiloride block of acidosis-induced currents of hASIC3-expressing HEK293 cells are shown.

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Cardiomyocytes – “Recordings of action potentials in mouse ES cell-derived Cor.At cardiomyocytes on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Axiogenesis.  

Cor.At® cardiomyocytes display typical cardiac ion channel activity and action potentials. Both the hERG blockers Quinidine and Cisapride, and the Na+-channel blocker Lidocaine, modulated the action potentials. The Quinidine and Lidocaine effects were reversible, the Cisapride effect was non-reversible. The results demonstrate the presence of an array of ion channels in Cor.At® cardiomyocytes which in conjunction are capable of generating action potentials. Hence these stem cell-derived cardiomyocytes are a suitable alternative to primary cardiomyocytes in drug screening and safety testing. In addition, these experiments demonstrate for the first time the suitability of a higher throughput planar patch clamp system, i.e. Nanion´s Patchliner®, for recording action potentials. This is possible because of the flexibility of Nanion‘s patch clamp systems allowing for a multitude of different experiments to be performed in both voltage and current clamp modes. Cells expressing multiple ion channels and therefore able to elicit action potentials, as opposed to cell lines overexpressing a single ion channel subtype, better represent the physiological system. Thus, the Patchliner® in combination with Cor.At® cardiomyocytes form a powerful tool for ion channel research, drug screening and safety testing.

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Acetylcholine Receptor Alpha 7 – “Human a7 nicotinic Acetylcholine Receptor on the Patchliner “
Patchliner application note:   Cells were kindly provided by Galantos.

The human a7 nicotinic acetylcholine receptor expressing cells (stable) from Galantos Pharma GmbH were tested on the Patchliner. Repeated stimulations with 100 μM nicotine were tested to validate the system. In a run, six out of eight cells could be measured. All six cells showed characteristic a7 currents. Average peak currents for the shown experiment were 0.9 ± 0.2 nA (n = 6) which is consistent with manual patch clamp results reported by Galantos Pharma.

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AMPA receptor (GluA2) – “Activation, potentiation and inhibition of AMPA receptors on the Patchliner”
Patchliner application note:

AMPA receptors are cation-permeable ionotropic glutamate receptors of the non-NMDA receptor subfamily. To date four subunits, GluA1-4, have been identified which are of similar size (approx. 900 kDa) and share 68-73% amino acid sequence identity. The human GluA2 subunit is encoded by the GRIA2 gene located on the 4q32-33 chromosome. Each of the 4 subunits has four distinct domains: an extracellular amino acid terminal domain (ATD); the extracellular ligand binding domain (LBD); the transmembrane domain (TMD) with 3 transmembrane segments (M1, M3 and M4) and 1 cytoplasmic facing re-entrant loop (M2); and an intracellular carboxyterminal domain. The functional receptor exists as a tetramer, either as homomers or heteromers (GluA1 and GluA4). The vast majority of excitatory fast synaptic transmission in the mammalian central nervous system is mediated by AMPA receptors of differing subunit combinations. It is well known that glutamate is a neurotoxin and it is proposed that overactivation of ionotropic glutamate receptors may underlie many neurodegenerative disorders such as ischemic stroke, epilepsy, Parkinson’s and dementia, amongst others. Enhancement of AMPA receptor activation by, for example, BDNF, has been proposed to have beneficial effects on learning and memory and has potential therapeutic value in the treatment of depression, Huntington’s and  Parkinson’s diseases. Here we present data collected on an 8-channel Patchliner showing recordings of GluA2-mediated currents. Glutamate activated GluA2 receptors with an EC50 similar to those reported in the literature. CNQX inhibited and LY404187 enhanced GluA2-mediated responses.

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NaV1.5-Late – “INa-Late recorded from CHO cells and hiPSC-CMs on Nanion´s Patchliner”
Patchliner application note:   Cells were kindly provided by Charles River. iCell® Cardiomyocytes2 were kindly provided by Fujifilm Cellular Dynamics International.

The voltage-gated Na+ channel 1.5 (NaV1.5) is encoded by the SCN5A gene and is responsible for the rising phase of the cardiac action potential (AP). The NaV1.5 channel is comprised of a pore-forming α subunit and auxillary β subunits. When the cardiac cell membrane depolarizes, NaV1.5 opens for a short time allowing an influx of Na+ ions resulting in the upstroke of the AP. During the AP, these channels can recover from inactivation and re-open resulting in a sustained current termed INa-Late. Although this current is substantially smaller than the peak Na+ current (INa-Peak), it is active during the plateau phase and therefore contributes to AP morphology. There is a growing body of evidence that increased INa-Late can have a pathophysiological role in acquired heart diseases such as myocardial ischemia and heart failure. INa-Late is elevated in several pathological conditions which could result in Na+-overload in these cells. A number of loss or gain-of-function mutations in the SCN5A gene have been identified which lead to changes in the magnitude or duration of INa-Peak or INa-Late resulting in fatal arrhythmias. INa-Late is a potential drug target to treat cardiac disorders such as angina, heart failure and arrhythmia. It is also an important target in safety pharmacology as enhancement of INa-Late is proarrhythmic.In this study the Patchliner was used to record INa-Late from CHO cells and hiPSC-CMs. INa-Late was recorded using the voltage protocol specified by CiPA and activated using ATX-II. INa-Late could be recorded from CHO cells stably expressing NaV1.5 and blocked by lidocaine. INa-Late could also be detected in iCell® Cardiomyoctes2 and blocked by ranolazine.

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Acetylcholine Receptor Alpha 3 Beta 4 – ” Nicotinic a3b4 receptors recorded on Nanion’s Patchliner”
Patchliner application note

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel superfamily which includes GABAA, 5HT3, NMDA and glycine receptors. It is a cation-permeable ion channel activated by the neurotransmitter acetylcholine and the natural alkaloid, nicotine. Neuronal nAChR are pentameric and functional channels are formed from a repertoire of nine α (α2 to α10) and three β subunits (β2 to β4). Most nAChR exist as heteromers with the stoichiometry 2α to 3β, however some α subunits function as homomers, these being α7 or α9. nAChR have been proposed to play a role in many neurological disorders such as Alzheimer’s Disease, Parkinson’s, Tourette’s Syndrome and depression. mRNA for the α3 and β4 subunits is found in the mammalian CNS and PNS, in particular automomic ganglion cells and chromaffin cells. nAChR containing the α3 subunit are essential for mediating fast synaptic  transmission in the autonomic nervous system and is essential for survival. Block of nicotinic α3β4 receptors by methadone has also been suggested to play a potential role in analgesia. Here we present data collected on a 4- or 8-channel Patchliner showing the potential use of the Patchliner to record nAChR α3β4 currents activated by nicotine. Nicotine activated α3β4 receptors in a concentration dependent manner with an EC50 similar to those reported in the literature. Nicotinic α3β4 receptors could be repetitively activated by nicotine and blocked by mecamylamine, a ganglionic blocker with clinically relevant hypotensive actions, with an IC50 in good agreement with the literature.

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NaV1.5 – “Characterization of CreaCell’s hNaV1.5 (A-0822) on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by CreaCell.

In this Application Note we present data characterizing hNaV1.5 overexpressing HEK293 cells. The data were collected with Nanion‘s Patchliner. The performance of the cells was very good on the Patchliner. Current responses of an individual cell expressing the hNaV1.5 channel to an IV voltage protocol are shown as well as the corresponding current-voltage relationship. The average mean peak current density of the cells was -215.1 ± 63 pA/pF at 0 mV (n=9).

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NaV1.5 – “Pharmacology of hNaV1.5 recorded on Nanion’s Patchliner”
Patchliner application note:   Cells were kindly provided by Millipore.

Voltage gated sodium channels (NaV) are important elements of action potential initiation and propagation in excitable cells. The channels are activated upon a depolarization of the membrane. Their activation leads to further depolarization of the membrane which constitutes the upstroke of the action potential. NaV currents generally activate very fast (within 1-2 ms) upon depolarization of the membrane. Hence, a good and stable access resistance is critical for high quality pharmacological patch clamp recordings. Also, for automated patch clamp devices, it is not a given that applied compound concentrations are accurately delivered to the cell. This is a pre-requisite for accurately reproducible dose-response curves. Here we present data collected on the 8-channel Patchliner. Tetrodotoxin and lidocaine dose‑response curves on hNaV1.5 expressed in HEK293 cells are shown. Lidocaine has been shown to block hNaV1.5 in its inactivated state (Bean et al. 1983) which means that the IC50 of lidocaine becomes dependent on the holding potential. This dependence was investigated. We also demonstrate the stability and reproducibility of the data collected with the Patchliner. Using two sequential dose responses of hNaV1.5 to TTX we demonstrate that the compound concentrations are accurately delivered to the cells and that recordings are stable with robust access resistance.

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2022 – Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider
Patchliner Publication in PNAS (2022) Authors: Finol-Urdaneta R. K., Ziegman R., Dekan Z., McArthur J. R., Heitmann S., Luna-Ramirez K., Tae H-S., Mueller A., Starobova H., Chin Y. K.-Y., Wingerd J. S., Undheim E. A. B., Cristofori-Armstrong B., Hill A. P., Herzig V., King G. F., Vetter I., Rash L. D., Adams D. J., Alewood P. F.

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.

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2022 – Synergistic Adverse Effects of Azithromycin and Hydroxychloroquine on Human Cardiomyocytes at a Clinically Relevant Treatment Duration
Patchliner Publication in Pharmaceuticals (2022) Authors: Li W., Luo X., Poetsch M. S., Oertel R., Nichani K., Schneider M., Strano A., Hasse M., Steiner R-P., Cyganek L., Hettwer K., Uhlig S., Simon K., Guan K., Schubert M.

Adverse effects of drug combinations and their underlying mechanisms are highly relevant for safety evaluation, but often not fully studied. Hydroxychloroquine (HCQ) and azithromycin (AZM) were used as a combination therapy in the treatment of COVID-19 patients at the beginning of the pandemic, leading to higher complication rates in comparison to respective monotherapies. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ, AZM, and their combination on the structure and functionality of cardiomyocytes, and to better understand the underlying mechanisms. Our results demonstrate synergistic adverse effects of AZM and HCQ on electrophysiological and contractile function of iPSC-CMs. HCQ-induced prolongation of field potential duration (FPDc) was gradually increased during 7-day treatment period and was strongly enhanced by combination with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, more pronounced contractile dysfunctions, and more elevated conduction velocity, compared to respective monotreatments. Mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased cellular accumulation of HCQ and AZM as well as increased Cx43- and NaV1.5-protein levels.

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2022 – Ion Channel Modeling beyond State of the Art: A Comparison with a System Theory-Based Model of the Shaker-Related Voltage-Gated Potassium Channel KV1.1
Patchliner Publication in Cells (2022) Authors: Langthaler S., Lozanović Šajić J., Rienmüller T., Weinberg S. H., Baumgartner C.

The mathematical modeling of ion channel kinetics is an important tool for studying the electrophysiological mechanisms of the nerves, heart, or cancer, from a single cell to an organ. Common approaches use either a Hodgkin–Huxley (HH) or a hidden Markov model (HMM) description, depending on the level of detail of the functionality and structural changes of the underlying channel gating, and taking into account the computational effort for model simulations. Here, we introduce for the first time a novel system theory-based approach for ion channel modeling based on the concept of transfer function characterization, without a priori knowledge of the biological system, using patch clamp measurements. Using the shaker-related voltage-gated potassium channel KV1.1 (KCNA1) as an example, we compare the established approaches, HH and HMM, with the system theory-based concept in terms of model accuracy, computational effort, the degree of electrophysiological interpretability, and methodological limitations. This highly data-driven modeling concept offers a new opportunity for the phenomenological kinetic modeling of ion channels, exhibiting exceptional accuracy and computational efficiency compared to the conventional methods. The method has a high potential to further improve the quality and computational performance of complex cell and organ model simulations, and could provide a valuable new tool in the field of next-generation in silico electrophysiology.

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2022 – Low potency inhibition of NaV1.7 by externally applied QX-314 via a depolarizing shift in the voltage-dependence of activation
Patchliner Publication in European Journal of Pharmacology (2022) Authors: Klasfauseweh T., Israel M.R., Ragnarsson L., Cox J.J., Durek T., Carter D.A., Leffler A., Vetter I., Deuis J.R.

QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.

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2022 – Discovery of SHR5133, a Highly Potent and Novel HBV Capsid Assembly Modulator
Patchliner Publication in ACS Medicinal Chemistry Letters (2022) Authors: Li X., Zhang Z., Chen Y., Wang B., Yang G., Xu X., Yechao B., Bai D., Feng B., Mao Y., Feng J., Bai C., He F., Tao W.

Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.

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2022 – Identification of a sensory neuron CaV2.3 inhibitor within a new superfamily of macro-conotoxins
Patchliner Pre-Print Publication in bioRxiv (2022) Authors: Hackney C., Salcedo P., Mueller E., Kjelgaard L., Watkins M., Zachariassen L., McArthur J., Adams D., Kristensen A., Olivera B., Finol-Urdaneta R., Safavi-Hemami H., Morth J., Ellgaard L.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, that defines a new gene superfamily. The crystal structure of recombinant Mu8.1 shows structural similarity with conotoxins of the con-ikot-ikot superfamily, with both toxins displaying a saposin-like fold. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of voltage-gated ion channels, Mu8.1 preferentially inhibited the R-type (CaV2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of CaV2.3 and voltage-gated K+ (KV4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing CaV2.3. Importantly, this multidisciplinary study demonstrates the feasibility of large, disulfide-rich venom-component investigation, an endeavor that will lead to the discovery of novel structures and functions in the previously underexplored group of macro-conotoxins.

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2022 – Differentiating the Neuropharmacological Properties of Nicotinic Acetylcholine Receptor-Activating Alkaloids
Patchliner Publication in Frontiers in Pharmacology (2022) Authors: Alijevic O., Jaka O., Alzualde A., Maradze D., Xia W., Frentzel S., Gifford A., Peitsch M., Hoeng J., Koshibu K.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids—nicotine, cotinine, and anatabine—in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release—the neurotransmitter systems relevant for anxiety—were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4β2 and α6/3β2β3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4β2 and α6/3β2β3 subtypes at high doses and no detectable activities against α3β4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

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2022 – Discovery of agonist–antagonist pairs for the modulation of Ca [2]+ and voltage-gated K+ channels of large conductance that contain beta1 subunits
Patchliner Publication in Bioorganic & Medicinal Chemistry (2022) Authors: Slayden A.V., Dyer C.L., Ma D., Li W., Bukiya A.N., Parrill A.L., Dopico A.M.

Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.

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2022 – An in silico–in vitro pipeline for drug cardiotoxicity screening identifies ionic pro-arrhythmia mechanisms
Patchliner Publication in British Journal of Pharmacology (2022) Authors: Clark A., Wei S., Kalola D., Krogh-Madsen T., Christini D.

Background and PurposeBefore advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic.Experimental ApproachAn in silico–in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug.Key ResultsSignificant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IKr (a source of arrhythmias) by all strong IKr blockers, including cisapride, quinidine and quinine. The protocol also detected block of ICaL by verapamil and Ito by quinidine. Further demonstrating the power of the approach, the VC data uncovered a previously unidentified If block by quinine, which was confirmed with experiments using a HEK-293 expression system and automated patch-clamp.Conclusion and ImplicationsWe developed an in silico–in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening.

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2022 – Anticancer effect of verteporfin on non-small cell lung cancer via downregulation of ANO1
Patchliner Publication in Biomedicine & Pharmacotherapy (2022) Authors: Jeong S.B., Das R., Kim DH., Lee S., Oh H.I., Jo S., Lee Y., Kim J., Park S.J., Choi D.K., Moon U.K., Kwon OB., Namkung W., Lee S., Cho B.C., Woo J., Seo Y.

Anoctamin 1 (ANO1) is a calcium-activated chloride channel found in various cell types and is overexpressed in non-small cell lung cancer (NSCLC), a major cause of cancer-related mortality. With the rising interest in development of druggable compounds for NSCLC, there has been a corresponding rise in interest in ANO1, a novel drug target for NSCLC. However, as ANO1 inhibitors that have been discovered simultaneously exhibit both the functions of an inhibition of ANO1 channel as well as a reduction of ANO1 protein levels, it is unclear which of the two functions directly causes the anticancer effect. In this study, verteporfin, a chemical compound that reduces ANO1 protein levels was identified through high-throughput screening. Verteporfin did not inhibit ANO1-induced chloride secretion but reduced ANO1 protein levels in a dose-dependent manner with an IC50 value of ~300 nM. Moreover, verteporfin inhibited neither P2Y receptor-induced intracellular Ca2+ mobilization nor cystic fibrosis transmembrane conductance regulator (CFTR) channel activity, and molecular docking studies revealed that verteporfin bound to specific sites of ANO1 protein. Confirming that verteporfin reduces ANO1 protein levels, we then investigated the molecular mechanisms involved in its effect on NSCLC cells. Interestingly, verteporfin decreased ANO1 protein levels, the EGFR-STAT3 pathway as well as ANO1 mRNA expression. Verteporfin reduced the viability of ANO1-expressing cells (PC9, and gefitinib-resistant PC9) and induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage. However, it did not affect hERG channel activity. These results show that the anticancer mechanism of verteporfin is caused via the down-regulation of ANO1.

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2021 – The translocator protein ligands as mitochondrial functional modulators for the potential anti-Alzheimer agents
Patchliner Publication in Journal of Enzyme Inhibition and Medicinal Chemistry (2021) Authors: Kim T., Morshed M.N., Londhe A.M., Lim J.W., Lee H.E., Cho S., Cho S.J., Hwang H., Lim S.M., Lee J.Y., Lee J., Pae A.N.

Small molecule modulators of mitochondrial function have been attracted much attention in recent years due to their potential therapeutic applications for neurodegenerative diseases. The mitochondrial translocator protein (TSPO) is a promising target for such compounds, given its involvement in the formation of the mitochondrial permeability transition pore in response to mitochondrial stress. In this study, we performed a ligand-based pharmacophore design and virtual screening, and identified a potent hit compound, 7 (VH34) as a TSPO ligand. After validating its biological activity against amyloid-β (Aβ) induced mitochondrial dysfunction and in acute and transgenic Alzheimer’s disease (AD) model mice, we developed a library of analogs, and we found two most active compounds, 31 and 44, which restored the mitochondrial membrane potential, ATP production, and cell viability under Aβ-induced mitochondrial toxicity. These compounds recovered learning and memory function in acute AD model mice with improved pharmacokinetic properties.

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2022 – A nutraceutical product, extracted from Cannabis sativa, modulates voltage-gated sodium channel function
Patchliner Publication in Journal of Cannabis Research (2022) Authors: Milligan C.J., Anderson L.L., Bowen M.T., Banister S.D., McGregor I.S., Arnold J.C., Petrou S.

Purified cannabidiol (CBD), a non-psychoactive phytocannabinoid, has gained regulatory approval to treat intractable childhood epilepsies. Despite this, artisanal and commercial CBD-dominant hemp-based products continue to be used by epilepsy patients. Notably, the CBD doses used in these latter products are much lower than that found to be effective in reducing seizures in clinical trials with purified CBD. This might be because these CBD-dominant hemp products contain other bioactive compounds, including phytocannabinoids and terpenes, which may exert unique effects on epilepsy-relevant drug targets. Voltage-gated sodium (NaV) channels are vital for initiation of neuronal action potential propagation and genetic mutations in these channels result in epilepsy phenotypes. Recent studies suggest that NaV channels are inhibited by purified CBD. However, the effect of cannabis-based products on the function of NaV channels is unknown.

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2021 – The Agonist Action of Alkylphenols on TRPA1 Relates to Their Effects on Membrane Lipid Order: Implications for TRPA1-Mediated Chemosensation
Patchliner Publication in International Journal of Molecular Sciences (2021) Authors: Startek J.B., Milici A., Naert R., Segal A., Alpizar Y.A., Voets T., Talavera K.

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds.

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2021 – The rescue of F508del-CFTR by elexacaftor/tezacaftor/ivacaftor (Trikafta) in human airway epithelial cells is underestimated due to the presence of ivacaftor
Patchliner Publication in European Respiratory Journal (2021) Authors: Becq F., Mirval S., Carrez T., Lévêque M., Billet A., Coraux C., Sage E., Cantereau A.

Trikafta, currently the leading therapeutic in Cystic Fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch clamp recordings, Western blot and immunolocalization experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and non-treated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor unlike the CFTR potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.

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2021 – Modeling of ion channels – A side by side comparison between Hodgkin Huxley and hidden Markov approach on the example of KV1.1
Patchliner Publication in Verlag der Technischen Universität Graz (2021) Authors: Langthaler S., Rienmüller T. M., Lozanovic Sajic J., Baumgartner C.

Mathematical models of individual ion channels form the building blocks of complex in-silico tools, enabling the investigation of biophysical mechanisms and simulation of disease processes. We here propose a first simplified hidden Markov Model (HMM) for the voltage-gated potassium channel KV1.1, taking into account the channels’ specific activation and inactivation characteristics close to physiological temperature. The modeling approach and simulation results were compared with an existing Hodgkin Huxley model based on the same experimental data. The newly developed HMM shows a higher accuracy with regard to the activation and inactivation behavior com- pared to the Hodgkin Huxley approach.

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2021 – Suppressing KV1.3 Ion Channel Activity with a Novel Small Molecule Inhibitor Ameliorates Inflammation in a Humanised Mouse Model of Ulcerative Colitis
Patchliner Publication in Journal of Crohn's and Colitis (2021) Authors: Unterweger A-L., Jensen M. Ø., Giordanetto F., Jogini V., Rüschher A., Seuß M., Winkelmann P., Koletzko L., Shaw D. E., Siebeck M., Gropp R., Beigel F., Aszodi A.

Background and Aims: The potassium channel KV1.3 is a potentially attractive therapeutic target in T cell-mediated inflammatory diseases, as the activity of antigen-activated T cells is selectively impeded by KV1.3 inhibition. In this study, we examined KV1.3 as a potential therapeutic intervention point for ulcerative colitis [UC], and studied the efficacy of DES1, a small-molecule inhibitor of KV1.3, in vitro and in vivo.Methods: KV1.3 expression on T cells in peripheral blood mononuclear cells [PBMCs] isolated from donors with and without UC was examined by flow cytometry. In biopsies from UC patients, KV1.3-expressing CD4+ T cells were detected by flow cytometry and immunohistochemistry. In vitro, we determined the ability of DES1 to inhibit anti-CD3-driven activation of T cells. In vivo, the efficacy of DES1 was determined in a humanised mouse model of UC and compared with infliximab and tofacitinib in head-to-head studies.Results: KV1.3 expression was elevated in PBMCs from UC patients and correlated with the prevalence of TH1 and TH2 T cells. KV1.3 expression was also detected on T cells from biopsies of UC patients. In vitro, DES1 suppressed anti-CD3-driven activation of T cells in a concentration-dependent manner. In vivo, DES1 significantly ameliorated inflammation in the UC model and most effectively so when PBMCs from donors with higher levels of activated T cells were selected for reconstitution. The efficacy of DES1 was comparable to that of either infliximab or tofacitinib.Conclusion: Inhibition of KV1.3 [by DES1, for instance] appears to be a potential therapeutic intervention strategy for UC patients.

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2021 – Effects of Hydroxychloroquine and Azithromycin on iPSC-derived Cardiomyocytes: Considerations for the Treatment of COVID-19 Patients
Patchliner Pre-Print Publication in bioRxiv (2021) Authors: Li W., Luo X., Poetsch M. S., Oertel R., Nichani K., Schneider M., Strano A., Hasse M., Steiner R-P., Cyganek L., Hettwer K., Uhlig S., Simon K., Guan K., Schubert M.

Despite known adverse effects of hydroxychloroquine (HCQ) and azithromycin (AZM) on cardiac function, HCQ and AZM have been used as combination therapy in the treatment of COVID-19 patients. Recent clinical data indicate higher complication rates with HCQ/AZM combination treatment in comparison to monotherapy. Here, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to systematically investigate the effects of HCQ and AZM individually and in combination. The clinically observed QT prolongation caused by treatment with HCQ could be recapitulated in iPSC-CMs based on prolonged field potential duration (FPDc). Interestingly, HCQ-induced FPDc prolongation was strongly enhanced by combined treatment with AZM, although AZM alone slightly shortened FPDc in iPSC-CMs. Furthermore, combined treatment with AZM and HCQ leads to higher cardiotoxicity, more severe structural disarrangement, and more pronounced contractile and electrophysiological dysfunctions, compared to respective mono-treatments. First mechanistic insights underlying the synergistic effects of AZM and HCQ on iPSC-CM functionality are provided based on increased Cx43- and NaV1.5-protein levels. Taken together, our results highlight that combined treatment with HCQ and AZM strongly enhances the adverse effects on cardiomyocytes, providing mechanistic evidence for the high mortality in patients receiving HCQ/AZM combination treatment.

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2021 – Effects of nicotinic acetylcholine receptor‑activating alkaloids on anxiety‑like behavior in zebrafish
 Patchliner Publication in Journal of Natural Medicines (2021) Authors: Alzualde A., Jaka O., Latino D.A.R.S., Alijevic O., Iturria I., Hurtado de Mendoza J., Pospisil P., Frentzel S., Peitsch M.C., Hoeng J., Koshibu K.

Alkaloids are a structurally complex group of natural products that have a diverse range of biological activities and significant therapeutic applications. In this study, we examined the acute, anxiolytic-like effects of nicotinic acetylcholine receptor (nAChR)-activating alkaloids with reported neuropharmacological effects but whose effects on anxiety are less well understood. Because α4β2 nAChRs can regulate anxiety, we first demonstrated the functional activities of alkaloids on these receptors in vitro. Their effects on anxiety-like behavior in zebrafish were then examined using the zebrafish novel tank test (NTT). The NTT is a relatively high-throughput behavioral paradigm that takes advantage of the natural tendency of fish to dive down when stressed or anxious. We report for the first time that cotinine, anatabine, and methylanatabine may suppress this anxiety-driven zebrafish behavior after a single 20-min treatment. Effective concentrations of these alkaloids were well above the concentrations naturally found in plants and the concentrations needed to induce anxiolytic-like effect by nicotine. These alkaloids showed good receptor interactions at the α4β2 nAChR agonist site as demonstrated by in vitro binding and in silico docking model, although somewhat weaker than that for nicotine. Minimal or no significant effect of other compounds may have been due to low bioavailability of these compounds in the brain, which is supported by the in silico prediction of blood–brain barrier permeability. Taken together, our findings indicate that nicotine, although not risk-free, is the most potent anxiolytic-like alkaloid tested in this study, and other natural alkaloids may regulate anxiety as well.

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2021 – Discovery and Preclinical Characterization of Usmarapride (SUVN-D4010): A Potent, Selective 5-HT4 Receptor Partial Agonist for the Treatment of Cognitive Deficits Associated with Alzheimer’s Disease
Patchliner Publication in Journal of Medicinal Chemistry (2021) Authors: Nirogi R., Mohammed A. R., Shinde A. K., Gagginapally S. R., Kancharla D. M., Ravella S. R., Bogaraju N., Middekadi V. R., Subramanian R., Palacharla R. C., Benade V., Muddana N., Abraham R., Medapati R. B., Thentu J. B., Mekala V. R., Petlu S., Lingavarapu B. B., Yarra S., Kagita N., Goyal V. K., Pandey S. K., Jasti V.

A series of oxadiazole derivatives were synthesized and evaluated as 5-hydroxytryptamine-4 receptor (5-HT4R) partial agonists for the treatment of cognitive deficits associated with Alzheimer’s disease. Starting from a reported 5-HT4R antagonist, a systematic structure–activity relationship was conducted, which led to the discovery of potent and selective 5-HT4R partial agonist 1-isopropyl-3-{5-[1-(3-methoxypropyl) piperidin-4-yl]-[1,3,4]oxadiazol-2-yl}-1H-indazole oxalate (Usmarapride, 12l). It showed balanced physicochemical–pharmacokinetic properties with robust nonclinical efficacy in cognition models. It also showed disease-modifying potential, as it increased neuroprotective soluble amyloid precursor protein alpha levels, and dose-dependent target engagement and correlation of efficacy with oral exposures. Phase 1 clinical studies have been completed and projected efficacious concentration was achieved without any major safety concerns. Phase 2 enabling long-term safety studies have been completed with no concerns for further development.

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2021 – Dynamic Clamp in Electrophysiological Studies on Stem Cell–Derived Cardiomyocytes—Why and How?
Patchliner Review Article in Journal of Cardiovascular Pharmacology (2021) Authors: Verkerk A.O., Wilders R.

Human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) are supposed to be a good human-based model, with virtually unlimited cell source, for studies on mechanisms underlying cardiac development and cardiac diseases, and for identification of drug targets. However, a major drawback of hPSC-CMs as a model system, especially for electrophysiological studies, is their depolarized state and associated spontaneous electrical activity. Various approaches are used to overcome this drawback, including the injection of “synthetic” inward rectifier potassium current (IK1), which is computed in real time, based on the recorded membrane potential(“dynamic clamp”). Such injection of an IK1-like current results in quiescent hPSC-CMs with a nondepolarized resting potential that show “adult-like” action potentials on stimulation, with functional availability of the most important ion channels involved in cardiac electrophysiology.These days, dynamic clamp has become a widely appreciated electrophysiological tool. However, setting up a dynamic clamp system can still be laborious and difficult, both because of the required hardware and the implementation of the dedicated software.In the present review, we first summarize the potential mechanisms underlying the depolarized state of hPSC-CMs and the functional consequences of this depolarized state. Next, we explain how an existing manual patch clamp setup can be extended with dynamic clamp. Finally, we shortly validate the extended setup with atrial-like and ventricular-like hPSC-CMs. We feel that dynamic clamp is a highly valuable tool in the field of cellular electrophysiological studies on hPSC-CMs and hope that our directions for setting up such dynamic clamp system may prove helpful.

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2021 – Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry
Patchliner Publication in Biotechnology Letters (2021) Authors: Shekdar K., Langer J., Venkatachalan S., Schmid L., Anobile J., Shah P., Lancaster A., Babich O., Dedova O., Sawchuck D.

Objective: Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.Methods: The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.Results: Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

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2021 – Discovery and Characterization of the Potent and Highly Selective 1,7-Naphthyridine-Based Inhibitors BAY-091 and BAY-297 of the Kinase PIP4K2A
Patchliner Publication in Journal of Medicinal Chemistry (2021) Authors: Wortmann L., Bräuer N., Holton S.J., Irlbacher H., Weiske J., Lechner C., Meier R., Karén J., Siöberg C.B., Pütter V., Christ C.D., ter Laak A., Lienau P., Lesche R., Nicke B., Cheung S-H., Bauser M., Haegebarth A., von Nussbaum F., Mumberg D., Lemos C.

PIP4K2A is an insufficiently studied type II lipid kinase that catalyzes the conversion of phosphatidylinositol-5-phosphate (PI5P) into phosphatidylinositol 4,5-bisphosphate (PI4,5P2). The involvement of PIP4K2A/B in cancer has been suggested, particularly in the context of p53 mutant/null tumors. PIP4K2A/B depletion has been shown to induce tumor growth inhibition, possibly due to hyperactivation of AKT and reactive oxygen species-mediated apoptosis. Herein, we report the identification of the novel potent and highly selective inhibitors BAY-091 and BAY-297 of the kinase PIP4K2A by high-throughput screening and subsequent structure-based optimization. Cellular target engagement of BAY-091 and BAY-297 was demonstrated using cellular thermal shift assay technology. However, inhibition of PIP4K2A with BAY-091 or BAY-297 did not translate into the hypothesized mode of action and antiproliferative activity in p53-deficient tumor cells. Therefore, BAY-091 and BAY-297 serve as valuable chemical probes to study PIP4K2A signaling and its involvement in pathophysiological conditions such as cancer.

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2021 – BAY-8400: A Novel Potent and Selective DNA-PK Inhibitor which Shows Synergistic Efficacy in Combination with Targeted Alpha Therapies
Patchliner Publication in Journal of Medicinal Chemistry (2021) Authors: Berger M., Wortmann L., Buchgraber P., Lücking U., Zitzmann-Kolbe S., Wengner A.M., Bader B., Bömer U., Briem H., Eis K., Rehwinkel H., Bartels F., Moosmayer D., Eberspächer U., Lienau P., Hammer S., Schatz C.A., Wang Q., Wang Q., Mumberg D., Nising C.F., Siemeister G.

Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.

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2021 – Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study
Patchliner Publication in Stem Cell Research (2021) Authors: Li W., Luo X., Ulbricht Y., Guan K.

Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 µM blebbistatin (BB, an excitation–contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput.

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2021 – A model-guided pipeline for drug cardiotoxicity screening with human stem-cell derived cardiomyocytes
Patchliner Pre-Print Publication in bioRxiv (2021) Authors: Clark A. P., Wie S., Krogh-Madsen T., Christini D. J.

New therapeutic compounds go through a preclinical drug cardiotoxicity screening process that is overly conservative and provides limited mechanistic insight, leading to the misclassification of potentially beneficial drugs as proarrhythmic. There is a need to develop a screening paradigm that maintains this high sensitivity, while ensuring non-cardiotoxic compounds pass this phase of the drug approval process. In this study, we develop an in vitro-in silico pipeline using human induced stem-cell derived cardiomyocytes (iPSC-CMs) to address this problem. The pipeline includes a model-guided optimization that produces a voltage-clamp (VC) protocol to determine drug block of seven cardiac ion channels. Such VC data, along with action potential (AP) recordings, were acquired from iPSC-CMs before and after treatment with a control solution or a low-, intermediate-, or high-risk drug. We identified significant AP prolongation (a proarrhythmia indicator) in two high-risk drugs and, from the VC data, determined strong ion channel blocks that led to the AP changes. The VC data also uncovered an undocumented funny current (If) block by quinine, which we confirmed with experiments using a HEK-293 expression line. We present a new approach to cardiotoxicity screening that simultaneously evaluates proarrhythmia risk (e.g. AP prolongation) and mechanism (e.g. channel block) from iPSC-CMs.

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2021 – Antiarrhythmic Hit to Lead Refinement in a Dish Using Patient-Derived iPSC Cardiomyocytes
Patchliner Publication in Journal of Medicinal Chemistry Authors: Cashman J.R., Ryan D., McKeithan W.L., Okolotowicz K., Gomez-Galeno J., Johnson M., Sampson K.J., Kass R.S, Pezhouman A, Karagueuzian H.S., Mercola M.,

Ventricular cardiac arrhythmia (VA) arises in acquired or congenital heart disease. Long QT syndrome type-3 (LQT3) is a congenital form of VA caused by cardiac sodium channel (INaL) SCN5A mutations that prolongs cardiac action potential (AP) and enhances INaL current. Mexiletine inhibits INaL and shortens the QT interval in LQT3 patients. Above therapeutic doses, mexiletine prolongs the cardiac AP. We explored structure-activity relationships (SAR) for AP shortening and prolongation using dynamic medicinal chemistry and AP kinetics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using patient-derived LQT3 and healthy hiPSC-CMs, we resolved distinct SAR for AP shortening and prolongation effects in mexiletine analogues and synthesized new analogues with enhanced potency and selectivity for INaL. This resulted in compounds with decreased AP prolongation effects, increased metabolic stability, increased INaL selectivity, and decreased avidity for the potassium channel. This study highlights using hiPSC-CMs to guide medicinal chemistry and "drug development in a dish".

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2020 – Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells
Patchliner Publication in Cell Communication and Signaling (2020) Authors: Bissinger R., Petkova-Kirova P., Mykhailova O., Oldenborg PA., Novikova E., Donkor D.A., Dietz T., Bhuyan A.A.M., Sheffield W.P., Grau M., Artunc F., Kaestner L., Acker J.P., Qadri S.M.

Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs).

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2020 – Utilising Automated Electrophysiological Platform in Epilepsy Research
Patchliner Chapter in Patch Clamp Electrophysiology (2020) Authors: Milligan C.J., Pachernegg S.

Chapter 7:Utilising Automated Electrophysiological Platform in Epilepsy ResearchAbstract:Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability.Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic.This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.

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2020 – Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity
Patchliner Publication in European Journal of Medicinal Chemistry (2020) Authors: Froux L., Elbahnsi A., Boucherle B., Billet A., Baatallah N., Hoffmann B., Alliot J., Zelli R., Zeinyeh W., Haudecoeur R., Chevalier B., Fortuné A., Mirval S., Simard C., Lehn P., Mornon J-P., Hinzpeter A., Becq F., Callebaut I., Décout J-L.

Recent evidence shows that combination of correctors and potentiators, such as the drug ivacaftor (VX-770), can significantly restore the functional expression of mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR), an anion channel which is mutated in cystic fibrosis (CF). The success of these combinatorial therapies highlights the necessity of identifying a broad panel of specific binding mode modulators, occupying several distinct binding sites at structural level. Here, we identified two small molecules, SBC040 and SBC219, which are two efficient cAMP-independent potentiators, acting at low concentration of forskolin with EC50 close to 1 μM and in a synergic way with the drug VX-770 on several CFTR mutants of classes II and III. Molecular dynamics simulations suggested potential SBC binding sites at the vicinity of ATP-binding sites, distinct from those currently proposed for VX-770, outlining SBC molecules as members of a new family of potentiators.

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2020 – The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations
Patchliner Publication in Nature Scientific Reports (2020) Authors: Orvos P., Pászti B., Topal L., Gazdag P., Prorok J., Polyák A., Kiss T., Tóth-Molnár E., Csupor-Löffler B., Bajtel A., Varró A., Hohmann J., Virág L., Csupor D.

Cannabis use is associated with cardiovascular adverse effects ranging from arrhythmias to sudden cardiac death. The exact mechanism of action behind these activities is unknown. The aim of our work was to study the effect of cannabidiol (CBD), tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol on cellular cardiac electrophysiological properties including ECG parameters, action potentials, hERG and IKr ion channels in HEK cell line and in rabbit and guinea pig cardiac preparations. CBD increased action potential duration in rabbit and guinea pig right ventricular papillary muscle at lower concentrations (1 µM, 2.5 µM and 5 µM) but did not significantly change it at 10 µM. CBD at high concentration (10 µM) decreased inward late sodium and L-type calcium currents as well. CBD inhibited hERG potassium channels with an IC50 value of 2.07 µM at room temperature and delayed rectifier potassium current with 6.5 µM at 37 °C, respectively. The frequency corrected QT interval (QTc) was significantly lengthened in anaesthetized guinea pig without significantly changing other ECG parameters. Although the IC50 value of CBD was higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that hERG and potassium channel inhibition might have a role in the possible proarrhythmic adverse effects of cannabinoids in situations where metabolism of CBD impaired and/or the repolarization reserve is weakened.

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2020 – Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators
Patchliner Publication in SLAS DISCOVERY (2020) Authors: Walsh, K. B.

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

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2020 – Structural basis of the potency and selectivity of Urotoxin, a potent KV1 blocker from scorpion venom
Patchliner Publication in Biochemical Pharmacology (2020) Authors: Luna-Ramirez K., Csoti A., McArthur J.R., Chin Y.K.Y., Anangi R., del Carmen Najera R., Possani L.D., King G.F., Panyi G., Yu H., Adams D.J., Finol-Urdaneta R.K.

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of KV1.2 described to date (IC50 = 160 pM). The native peptide also inhibits KV1.1, KV1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of KV1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/β (CSα/β) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against KV1.1–1.3 and KV1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all KV channels tested, without perturbing the toxin’s structure. Reciprocal mutations in the pore of Uro-sensitive KV1.2 and Uro-resistant KV1.5 channels revealed a direct interaction between Urotoxin and the KV channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its KV1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the KV1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of KV1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin.

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2020 – N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity
Patchliner Publication in Cancer Cell International (2020) Authors: Keenan B., Finol-Urdaneta R.K., Hope A., Bremner J.B., Kavallaris M., Lucena-Agell D., Ángela Oliva M., Díaz J.F & Vine K.L.

Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.

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2020 – Pharmacological and genetic characterisation of the canine P2X4 receptor
Patchliner Publication in British Journal of Pharmacology (2020) Authors: Sophocleous R.A., Berg, T., Finol‐Urdaneta R.K., Sluyter V., Keshiya S., Bell L., Curtis S.J., Curtis B.L., Seavers A., Bartlett R., Dowton M., Stokes L., Ooi L., Sluyter R.

Background and PurposeThe P2X4 receptor is an emerging therapeutic target for the treatment of chronic pain and cardiovascular disease. Dogs are well‐recognised natural models of human disease but information regarding P2X4 in dogs is absent. To aid the development and validation of P2X4‐targeting therapeutics, this study aimed to characterise and compare canine and human P2X4.Experimental ApproachGenomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. P2X4 pharmacology was characterised using nucleotide‐induced Fura‐2 AM measurements of intracellular Ca2+ responses and known P2X4 antagonists in 1321N1 and HEK293 cells. P2X4‐mediated inward currents in HEK293 cells were assessed by automated patch clamp.Key ResultsNo P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 were localised primarily to lysosomal compartments. ATP was identified as the primary agonist of canine P2X4 with near identical efficacy and potency at human P2X4. 2’(3’)‐O‐(4‐benzoyl)benzoyl ATP (BzATP), but not ADP, was identified as a partial agonist with reduced potency for dog P2X4 compared to the human orthologue. Five antagonists inhibited canine P2X4, with BX430 displaying reduced sensitivity and potency against canine P2X4.Conclusion and ImplicationsP2X4 is highly conserved across dog pedigrees and displays a similar expression pattern and pharmacological profile to human P2X4, providing support for validation and use of therapeutics designed for P2X4‐related disease onset and management in dogs and humans.

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2020 – Discovery and Development of SUVN-911: A Novel, Potent, Selective, and Orally Active Neuronal Nicotinic Acetylcholine α4β2 Receptor Antagonist for the Treatment of Depression
Patchliner Publication in Journal of Medicinal Chemistry (2020) Authors: Nirogi R., Mohammed A.R., Shinde A.K., Ravella S.R., Bogaraju N., Subramanian R., Mekala V.R., Palacharla R.C., Muddana N., Thentu J.B., Bhyrapuneni G., Abraham R., Jasti V.

A series of chemical optimizations guided by in vitro affinity at the α4β2 receptor in combination with selectivity against the α3β4 receptor, pharmacokinetic evaluation, and in vivo efficacy in a forced swim test resulted in identification of 3-(6-chloropyridine-3-yloxymethyl)-2-azabicyclo[3.1.0]hexane hydrochloride (9h, SUVN-911) as a clinical candidate. Compound 9h is a potent α4β2 receptor ligand with a Ki value of 1.5 nM. It showed >10 μM binding affinity toward the ganglionic α3β4 receptor apart from showing selectivity over 70 other targets. It is orally bioavailable and showed good brain penetration in rats. Marked antidepressant activity and dose-dependent receptor occupancy in rats support its potential therapeutic utility in the treatment of depression. It does not affect the locomotor activity at doses several folds higher than its efficacy dose. It is devoid of cardiovascular and gastrointestinal side effects. Successful long-term safety studies in animals and phase-1 evaluation in healthy humans for safety, tolerability, and pharmacokinetics paved the way for its further development.

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2020 – Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation
Patchliner Publication in Frontiers in Cell and Developmental Biology (2020) Authors: Li W., Stauske M., Luo X., Wagner S., Vollrath M., Mehnert C.S., Schubert M., Cyganek L., Chen S., Hasheminasab S.M., Wulf G., El-Armouche A., Maier L.S., Hasenfuss G., Guan K.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of INa density, a 69.5% reduction of NaV1.5 expression, and the impaired localization of NaV1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The Ito in BrS-CMs was significantly augmented, and the ICaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of Ito in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

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2020 – Assessment of the Effects of Online Linear Leak Current Compensation at Different Pacing Frequencies in a Dynamic Action Potential Clamp System
Patchliner Publication in Computing in Cardiology (2020) Authors: Fabbri A., Prins A., de Boer T.P.

Dynamic action potential (AP) clamp (dAPC) is an electrophysiology technique that allows one to study in real time the effects of a biological current included in a computational AP model. During an experiment, the seal resistance between the cell membrane and the pipette is finite and a leak current (Ileak) occurs. Its reduction is crucial to properly assess the effect of a drug. Our work aims to quantify the impact of Ileak on a ventricular AP model and to evaluate the benefits of an online compensation. We carried out the experiments using a prototype Nanion Patchliner Dynamite8 in dAPC mode, running the ten Tusscher human ventricular AP model. We use a passive model cell (Cm = 19.8pF, Rseal = 500MOhm) and online compensate the leak current adopting a linear model. Vmax, RMP and AP D20,50,90 are measured at several degrees of compensation (within 0 and 100%), at different pacing frequencies (0.5, 1, 2 Hz), and compared with the AP model in the open loop condition (i.e. with no connection between the model cell and the AP model). Ileak decreases Vmax, depolarizes RMP (up to +6.1mV at 1 Hz ) and prolongs AP D both during the plateau and the late repolarization; a full compensation of Ileak brings the AP biomarkers close to the open loop condition. With this test, we show that online compensation of Ileak is beneficial for proper assessment of AP biomarker. The correction of f RMP is key, as it affects the following phases of the AP.

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2020 – Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8
Patchliner and Dynamite8 Publication in Current Protocols in Pharmacology (2020) Authors: Becker N., Horváth A., De Boer T., Fabbri A., Grad C., Fertig N., George M., Obergrussberger A.

Current in vitro assays typically poorly predict cardiac liability as they focus on single ion channels overexpressed in cell lines. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs), on the other hand, provide a unique opportunity for drug testing on human cardiomyocytes using high‐throughput systems. However, these cells can differ from adult cardiomyocytes in their ion channel expression and, therefore, electrophysiologic properties. One of the main challenges of hiPSC‐CMs is the physiologic expression of ion channels such as the inward rectifiers (e.g., Kir2.1–2.3), which conduct the cardiac inward rectifier potassium current (IK1). IK1 is one of the primary contributors in maintaining a stable resting membrane potential in cardiac cells, which is essential for excitability. This is only expressed in low levels, or sometimes not at all, in hiPSC‐CMs as shown by patch clamp studies. Dynamic clamp is a method of electronically introducing ion currents (e.g., IK1) into cells to compensate for the lack of endogenous expression, thus offering the potential to record more stable action potentials in hiPSC‐CMs. In this article, we describe the method of using hiPSC‐CMs on an automated patch clamp device (Patchliner) coupled with the automated dynamic clamp add‐on (Dynamite8). We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc.Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell–derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistanceSupport Protocol 1: Cardiomyocyte plating and cultureSupport Protocol 2: Cell harvesting and dissociationAlternate Protocol: Recording action potential pharmacology at physiologic temperatures

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2020 – A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function
Patchliner Publication in The Faseb Journal (2020) Authors: Skarratt K.K., Gu B.J., Lovelace M.D., Milligan C.J., Stokes L., Glover R., Petrou S, Wiley J.S., Fuller S.J.

P2X7 is an ATP‐gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full‐length P2RX7A pre‐mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele‐specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C‐terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP‐binding site. P2X7L‐transfected HEK 293 cells have phagocytic but not channel, pore, or membrane‐blebbing function, and double‐transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP‐binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

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2020 – An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4β2 and α7 nicotinic acetylcholine receptors
Patchliner Publication in Phytochemistry (2020) Authors: Alijevic O., McHugh D., Rufener L., Mazurov A., Hoeng J., Peitsch M.

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4β2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4–7 Å are pharmacophoric elements necessary for molecular recognition by α4β2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids—nicotine, nornicotine, anabasine and R-anatabine—potently activated α4β2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4β2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4β2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4β2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4β2 and α7 nAChRs. It also revealed human α4β2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

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2019 – Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias
Patchliner Publication in Frontiers in Physiology (2019) Authors: Petkova-Kirova P., Hertz L., Danielczok J., Huisjes R., Makhro A., Bogdanova A., del Mar Mañú-Pereira M., Vives Corrons J.-L., van Wijk R., Kaestner L.

Congenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation.

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2020 – A novel pyrazolo [3,4-d] pyrimidine, KKC080106, activates the Nrf2 pathway and protects nigral dopaminergic neurons
Patchliner Publication in Experimental Neurology (2020) Authors: Lee J.A., Kim H.R., Son H.J., Shin N., Han S.H., Cheong C.S., Kim D.J., Hwanga O.

The transcription factor nuclear factor-erythroid 2-related factor-2 (Nrf2) is known to induce neuroprotective and anti-inflammatory effects and is considered to be an excellent molecular target for drugs related to neurodegenerative disease therapy. Nrf2 activators previously tested in clinical trials were electrophilic, causing adverse effects due to non-selective and covalent modification of cellular thiols. In order to circumvent this issue, we constructed and screened a chemical library consisting of 241 pyrazolo [3,4-d] pyrimidine derivatives and discovered a novel, non-electrophilic compound: 1-benzyl-6-(methylthio)-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine (KKC080106). KKC080106 was able to activate Nrf2 signaling as it increases the cellular levels of Nrf2, binds to the Nrf2 inhibitor protein Keap1, and causes the accumulation of nuclear Nrf2. We also observed an increase in the expression levels of Nrf2-dependent genes for antioxidative/neuroprotective enzymes in dopaminergic neuronal cells. In addition, in lipopolysaccharide-activated microglia, KKC080106 suppressed the generation of the proinflammatory markers, such as IL-1β, TNF-α, cyclooxygenase-2, inducible nitric oxide synthase, and nitric oxide, and inhibited the phosphorylation of kinases known to be involved in inflammatory signaling, such as IκB kinase, p38, JNK, and ERK. As a drug, KKC080106 exhibited excellent stability against plasma enzymes and a good safety profile, evidenced by no mortality after the administration of 2000 mg/kg body weight, and minimal inhibition of the hERG channel activity. Pharmacokinetic analysis revealed that KKC080106 has good bioavailability and enters the brain after oral and intravenous administration, in both rats and mice. In MPTP-treated mice that received KKC080106 orally, the compound blocked microglial activation, protected the nigral dopaminergic neurons from degeneration, and prevented development of the dopamine deficiency-related motor deficits. These results suggest that KKC080106 has therapeutic potential for neurodegenerative disorders such as Parkinson's disease.

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2019 – Isolation and Pharmacological Investigation of Compounds From Euphorbia matabelensis
Patchliner Publication in Natural Product Communications (2019) Authors: Hammadi R., Kúsz N., Mwangi P.W., Kulmány Á., Zupkó I., Orvos P., Tálosi L., Hohmann J., Vasas A.

This work deals with the isolation and pharmacological investigations of compounds of Euphorbia matabelensis. After multiple separation process, including thin layer chromatography (TLC), vacuum liquid chromatography, preparative TLC, and high-performance liquid chromatography, 1 diterpene (ingenol) and 2 flavonoids (naringenin and eriodictyol) were obtained from the methanol extracts prepared from the stems and roots of the plant. The structures of the isolated compounds were determined by nuclear magnetic resonance (NMR) and MS measurements and comparison with literature data. All compounds were isolated for the first time from the plant. Eriodictyol was detected for the first time from a Euphorbia species. The compounds were tested for their antiproliferative (on HeLa, C33a, MCF-7, and MDA-MB-231 cell lines) and GIRK channel blocking activities. None of the compounds proved to be active in these test systems..

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2019 – MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo
Patchliner Publication in Cell Stem Cell (2019) Authors: Fiedler L.R., Chapman K., Xie M., Maifoshie E., Jenkins M., Golforoush P.A., Bellahcene M., Noseda M., Faust D., Jarvis A., Newton G., Paiva M.A., Harada M., Stuckey D.J., Song W., Habib J., Narasimham P., Aqil R., Schneider M.D.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.

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2019 – GABRP regulates chemokine signalling, macrophage recruitment and tumour progression in pancreatic cancer through tuning KCNN4-mediated Ca2+ signalling in a GABA-independent manner
Patchliner Publication in Gut (2019) Authors: Jiang S.H., Zhu L.L., Zhang M., Li R.K., Yang Q., Yan J.Y., Zhang C., Yang J.Y., Dong F.Y., Dai M., Hu L.P., Li J., Li Q., Wang Y.H., Yang X.M., Zhang Y.L., Nie H.Z., Zhu L., Zhang X.L., Tian G.A., Zhang X.X., Cao X.Y., Tao L.Y., Huang S., Jiang Y.S., Hua R., Qian Luo K., Gu J.R., Sun Y.W., Hou S., Zhang Z.G.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. METHODS: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. RESULTS: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. CONCLUSIONS: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.

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2019 – GIRK channel activity of Hungarian mushrooms: From screening to biologically active metabolites
Patchliner Publication in Fitoterapia (2019) Authors: Ványolósa A., Orvos P., Chuluunbaatar B., Tálosi L., Hohmann J.

In the current study effects of fungal extracts on the G-protein-activated inwardly rectifying potassium channel (GIRK1/4) were screened using the automated patch-clamp method. 40 organic (n-hexane, chloroform, and 50% methanol) and aqueous extracts were prepared from 10 mushroom species native to Hungary. Among the examined fungal fractions of different polarities some n-hexane and chloroform extracts exerted considerable ion channel activity. One of the most active fungal species, Hypholoma lateritium was selected for further detailed examination to determine the compounds responsible for the observed pharmacological property. Evaluation of the ion channel activity of mushroom metabolites 1–10 revealed that lanosta-7,9(11)-diene-12β,21α-epoxy2α,3β,24β,25-tetraol (5) demonstrates remarkable blocking activity on GIRK current (IC50 395.1 ± 31.8 nM). Investigation of the selectivity of the GIRK inhibitory effect proved that lanosta-7,9(11)-diene-12β,21α-epoxy2α,3β,24β,25-tetraol (5) has only weak inhibitory activity on hERG channel (7.9 ± 2.8% at 100 μM), exerting more than three orders of magnitude lower blocking activity on hERG channel than on GIRK channel.

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2019 – Discovery of BNC375, a Potent, Selective, and Orally Available Type I Positive Allosteric Modulator of α7 nAChRs
Patchliner Publication in ACS Medicinal Chemistry Letters (2019) Authors: Harvey A.J., Avery T.D., Schaeffer L, Joseph C., Huff B.C., Singh R., Morice C., Giethlen B.,Grishin A.A., Coles C.J., Kolesik P., Wagner S., Andriambeloson E., Huyard B., Poiraud E., Paul D., O’Connor S.M.

Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure–kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure–activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.

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2019 – Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs
Patchliner and Dynamite8 Publication in Stem Cell Research (2019) Authors: Li W., Luo X., Y., Wagner M., Piorkowski C., El-Armouche A., Guan K.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have evolved into widely used and reliable cell sources for modeling cardiovascular channelopathies and for drug safety pharmacology. However, the electrophysiological and pharmacological applications of hiPSC-CMs are hampered by manual patch-clamp technique, which is labor-intensive and generates a low-output. The automated patch-clamp technique is showing potential to overcome this problem. Here, we describe a new dissociation method, with which we can harvest a vast number of single relaxed hiPSC-CMs with smooth membrane suited for automated patch-clamp. Using the automated whole-cell patch-clamp technology, we report a high success rate for cell capture and whole-cell access (around 70%). We are able to identify and record several currents and paced action potentials (APs) with different success rates, including Na+ current (INa), L-type Ca2+ current (ICaL), two specific K+ currents, the transient outward K+ current (Ito) and the inward rectifier K+ current (IK1). Moreover, we successfully applied dynamic current-clamp to virtually increase IK1 for AP recordings. Our study suggests that automated patch-clamp technology could be used to investigate the relevant ionic currents and APs in hiPSC-CMs. The combination of automated patch-clamp and hiPSC-CM technologies promises a wide range of applications in the future.

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2019 – Avoiding hERG-liability in drug design via synergetic combinations of different (Q)SAR methodologies and data sources: a case study in an industrial setting
Patchliner Publication in Journal of Cheminformatics (2019) Authors: Hanser T., Steinmetz F.P., Plante J., Rippmann F., and Krier M.

In this paper, we explore the impact of combining different in silico prediction approaches and data sources on the predictive performance of the resulting system. We use inhibition of the hERG ion channel target as the endpoint for this study as it constitutes a key safety concern in drug development and a potential cause of attrition. We will show that combining data sources can improve the relevance of the training set in regard of the target chemical space, leading to improved performance. Similarly we will demonstrate that combining multiple statistical models together, and with expert systems, can lead to positive synergistic effects when taking into account the confidence in the predictions of the merged systems. The best combinations analyzed display a good hERG predictivity. Finally, this work demonstrates the suitability of the SOHN methodology for building models in the context of receptor based endpoints like hERG inhibition when using the appropriate pharmacophoric descriptors.

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2019 – Discovery of a small molecule modulator of the KV1.1/KVβ1 channel complex that reduces neuronal excitability and in vitro epileptiform activity
Patchliner Publication in CNS Neuroscience & Therapeutics (2019) Authors: Niespodziany I., Mullier B., André V.M., Ghisdal P., Jnoff E., Moreno‐Delgado D., Swinnen D., Sands Z., Wood M., Wolff C.

Aims: KV1.1 (KCNA1) channels contribute to the control of neuronal excitability and have been associated with epilepsy. KV1.1 channels can associate with the cytoplasmic KVβ1 subunit resulting in rapid inactivating A‐type currents. We hypothesized that removal of channel inactivation, by modulating KV1.1/KVβ1 interaction with a small molecule, would lead to decreased neuronal excitability and anticonvulsant activity. Methods: We applied high‐throughput screening to identify ligands able to modulate the KV1.1‐T1 domain/KVβ1 protein complex. We then selected a compound that was characterized on recombinant KV1.1/KVβ1 channels by electrophysiology and further evaluated on sustained neuronal firing and on in vitro epileptiform activity using a high K+‐low Ca2+ model in hippocampal slices. Results: We identified a novel compound able to modulate the interaction of the KV1.1/KVβ1 complex and that produced a functional inhibition of KV1.1/KVβ1 channel inactivation. We demonstrated that this compound reduced the sustained repetitive firing in hippocampal neurons and was able to abolish the development of in vitro epileptiform activity. Conclusions: This study describes a rational drug discovery approach for the identification of novel ligands that inhibit KV1.1 channel inactivation and provides pharmacological evidence that such a mechanism translates into physiological effects by reducing in vitro epileptiform activity.

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2019 – A Kinetic Map of the Homomeric Voltage-Gated Potassium Channel (KV) Family
Patchliner Publication in Frontiers in Cellular Neuroscience (2019) Authors: Ranjan R., Logette E., Marani M., Herzog M., Tâche V., Scantamburlo E., Buchillier V., Markram H.

The voltage-gated potassium (KV) channels, encoded by 40 genes, repolarize all electrically excitable cells, including plant, cardiac, and neuronal cells. Although these genes were fully sequenced decades ago, a comprehensive kinetic characterization of all KV channels is still missing, especially near physiological temperature. Here, we present a standardized kinetic map of the 40 homomeric KV channels systematically characterized at 15, 25, and 35°C. Importantly, the KV kinetics at 35°C differ significantly from commonly reported kinetics, usually performed at room temperature. We observed voltage-dependent Q10 for all active KV channels and inherent heterogeneity in kinetics for some of them. Kinetic properties are consistent across different host cell lines and conserved across mouse, rat, and human. All electrophysiology data from all KV channels are made available through a public website (Channelpedia). This dataset provides a solid foundation for exploring kinetics of heteromeric channels, roles of auxiliary subunits, kinetic modulation, and for building accurate KV models.

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2019 – Automated Planar Patch-Clamp Recording of P2X Receptors
Patchliner book chapter in Ion Channels (2020) Authors: Milligan, C. J., Jiang L.H.

P2X receptors are a structurally and functionally distinctive family of ligand-gated ion channels that play important roles in mediating extracellular adenosine 5′-triphosphate (ATP) signaling in diverse physiological and pathophysiological processes. For several decades, the “manual” patch-clamp technique was regarded as the gold standard assay for investigating ion channel properties. More recently, breakthroughs in the development of automated patch-clamp technologies are enabling the study of ion channels, with much greater throughput capacities. These automated platforms, of which there are many, generate consistent, reliable, high-fidelity data. This chapter demonstrates the versatility of one of these technologies for ligand-gated ion channels, with a particular emphasis on protocols that address some of the issues of receptor desensitization that are commonly associated with P2X receptor-mediated currents.

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2018 – Evaluation of possible proarrhythmic potency: comparison of the effect of dofetilide, cisapride, sotalol, terfenadine and verapamil on hERG and native IKr currents and on cardiac action potential
Patchliner Publication in Toxicological Sciences (2018) Authors: Orvos P., Kohajda Z., Szlovák J., Gazdag P., Árpádffy-Lovas T., Tóth D., Geramipour A., Tálosi L., Jost N., Varró A., Virág L.

Introduction: The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by hERG test using automated patch-clamp systems in stable transfected cell lines. Aim of the present study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. Methods: The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Results: Dofetilide, cisapride, sotalol, terfenadine and verapamil blocked hERG channels at 37 °C with an IC50 of 7 nM, 18 nM, 343 μM, 165 nM and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37 °C were 13 nM, 26 nM, 52 μM, 54 nM and 268 nM, respectively. Cisapride, dofetilide and sotalol excessively lengthened, terfenadine and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Conclusions: Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.

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2018 – Selective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death
Patchliner Publication in PNAS (2018) Authors: Richards K.L., Milligan C.J., Richardson R.J., Jancovski N., Grunnet M., Jacobson L.H., Undheim E.A.B., Mobli M., Chow C.Y., Herzig V., Csoti A., Panyi G., Reid A.A., King G.F., Petrou S.

Significance: Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit NaV1.1. Cell-based assays showed Hm1a was selective for hNaV1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with NaV1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders. Abstract: Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.

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2018 – Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo
Patchliner Publication in Pharmacological Research (2018) Authors: Baburin I., Varkevisser R., Schramm A., Saxena P., Beyl S., Szkokan P., Linder T., Stary-Weinzinger A., van der Heyden M.A.G., Houtman M., Takanari H., Jonsson M., Beekman J.H.D., Hamburger M., Vos M.A., Hering S.

Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC-microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2 ± 26.3 nM and 144.8 ± 35.1 nM, respectively. In dog ventricular cardiomyocytes, DHE dose-dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10 μM DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (CaVB) dogs. In rabbits, DHE increased the QT interval significantly by 12 ± 10% (0.05 mg/kg/5 min) and 60 ± 26% (0.5 mg/kg/5 min), and induced Torsade de Pointes arrhythmias (TdP, 0.5 mg/kg/5 min) in 2 rabbits. In CaVB dogs, 0.33 mg/kg/5 min DHE increased QT duration by 48 ± 10% (P  0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), methanolic extracts of Evodia, DHE and hortiamine dose-dependently prolonged APD. At 3 μM DHE and hortiamine induced EADs.hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC-CMs and dose-dependent proarrhythmic effects of DHE at micromolar plasma concentrations in CaVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

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2018 – Electrophysiological investigation of the effect of structurally different bispyridinium non-oxime compounds on human α7-nicotinic acetylcholine receptor activity – An in vitro structure-activity analysis
Patchliner Publication in Toxicology Letters (2018) Authors: Scheffel C., Niessen K.V., Rappenglück S., Wanner K.T., Thiermann H., Worek F., Seeger T.

Organophosphorus compounds, including nerve agents and pesticides, exert their toxicity through irreversible inhibition of acetylcholinesterase (AChE) resulting in an accumulation of acetylcholine and functional impairment of muscarinic and nicotinic acetylcholine receptors. Current therapy comprises oximes to reactivate AChE and atropine to antagonize effects induced by muscarinic acetylcholine receptors. Nicotinic malfunction leading to depression of the central and peripheral respiratory system is not directly treated calling for alternative therapeutic interventions. In the present study, we investigated the electrophysiological properties of the human nAChR subtype α7 (hα7-nAChR) and the functional effect of the 4-tert-butyl bispyridinium (BP) compound MB327 and of a series of novel substituted bispyridinium compounds on the receptors by an automated patch clamp technique. Activation of hα7-nAChRs was induced by nicotine and acetylcholine demonstrating rapid cationic influx up to 100 μM. Agonist-induced currents decayed within a few milliseconds revealing fast desensitization of the receptors. Application of higher agonist concentrations led to a decline of current amplitudes which seemed to be due to increasing receptor desensitization. When 100 μM of agonist was coapplied with low concentrations of the well characterized α7-specific positive allosteric modulator PNU-120596 (1 μM–10 μM), the maximum response and duration of nAChR activation were markedly augmented indicating an elongated mean open-time of receptors and prevention of receptor desensitization. However, co-application of increasing PNU-120596 concentrations (>10 μM) with agonist induced a decline of potentiated current responses. Although less pronounced than PNU-120596, six of the twenty tested substituted BP compounds, in particular those with a substituent at 3-position and 4-position at the pyridinium moieties, were found to potentiate current responses of hα7-nAChRs, most pronounced MB327. This effect was clearly depended on the presence of the agonist indicating a positive allosteric mechanism of these compounds. Besides potentiation at low concentrations, these compounds seem to interact at different binding sites on hα7-nAChRs since enhancement decreased at high concentrations.

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2018 – A Previously Unrecognized Ca2+-inhibited Nonselective Cation Channel in Red Blood Cells
Patchliner Publication in HemaSphere (2018) Authors: Petkova-Kirova P., Hertz L., Makhro A., Danielczok J., Huisjes R., Llaudet-Planas E., Mañú-Pereira, M., Vives Corrons J.-L., van Wijk R., Bogdanova A., Kaestner L.

Storage lesion of red blood cells (RBCs) is a well-recognizedprocess characterized by complex morphological and functional changes. Those changes deteriorate the life-saving quality of stored blood with a reported increase in mortality for some categories of patients receiving “old” versus “new” blood. The importance of RBC cation gradients (K+and Na+) dissipation in the process of storage lesion has been recently highlighted. Here we report a previously unrecognized nonselective cation channel in human RBCs (patch-clamp) activated whenever extracellular Ca2+ is removed and very likely contributing to the cation gradients dissipation when opened. In view of the existence of such a channel the use of non-Ca2+-chelating anticoagulants like heparin, preventing channel opening, can reduce cation gradients dissipation and help limit and delay RBCs storage lesion.

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2018 – Counteracting desensitization of human α7-nicotinic acetylcholine receptors with bispyridinium compounds as an approach against organophosphorus poisoning
Patchliner Publication in Toxicology Letters (2018) Authors: Scheffel C., Niessen K.V., Rappenglück S., Wanner K.T., Thiermann H., Worek F., Seeger T.

Irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and overstimulation of muscarinic and nicotinic receptors accounts for the acute toxicity of organophosphorus compounds (OP). Accordingly, the mainstay pharmacotherapy against poisoning by OP comprises the competitive muscarinic acetylcholine receptor antagonist atropine to treat muscarinic effects and, in addition, oximes to reactivate inhibited AChE. A therapeutic gap still remains in the treatment of desensitized nicotinic acetylcholine receptors following OP exposure. Hereby, nicotinic effects result in paralysis of the central and peripheral respiratory system if untreated. Thus, these receptors pose an essential target for therapeutic indication to address these life-threatening nicotinic symptoms of the cholinergic crisis. Identification of ligands regulating dynamic transitions between functional states by binding to modulatory sites appears to be a promising strategy for therapeutic intervention. In this patch clamp study, the ability of differently substituted bispyridinium non-oximes to “resensitize” i.e. to recover the activity of desensitized human homomeric α7-type nAChRs stably transfected in CHO cells was investigated and compared to the already described α7-specific positive allosteric modulator PNU-120596. The structures of these bispyridinium analogues were based on the lead structure of the tert-butyl-substituted bispyridinium propane MB327, which has been shown to have a positive therapeutic effect due to a non-competitive antagonistic action at muscle-type nAChRs in vivo and has been found to have a positive allosteric activity at neuronal receptors in vitro. Prior to test compounds, desensitization of hα7-nAChRs was verified by applying an excess of nicotine revealing activation at low, and desensitization at high concentrations. Thereby, desensitization could be reduced by modulation with PNU-120596. Desensitization was further verified by dose-response profiles of agonists, carbamoylcholine and epibatidine in the absence and presence of PNU-120596. Although less pronounced than PNU-120596 and the lead structure MB327, bispyridinium compounds, particularly those substituted at position 3 and 4, resensitized the nicotine desensitized hα7-nAChRs in a concentration-dependent manner and prolonged the mean channel open time. In summary, identification of more potent compounds able to restore nAChR function in OP intoxication is needed for development of a putative efficient antidote.

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2017 – Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside
Patchliner Publication in Cellular Physiology and Biochemistry (2017) Authors: Ghashghaeinia M., Wesseling M.C., Ramos E., Petkova-Kirova P., Waibel S., Lang E., Bissinger R., Alzoubi K., Edelmann B., Hosseinzadeh Z., Dreischer P., Shahvaroughi-Farahani A., Mrowietz U., Köberle M., Kaestner L., Bernhardt I., Martínez-Ruiz A., Wieder T., Lang F.

Background and Purpose: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO.Methods:Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. Results: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. Conclusions: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

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2018 – A Hybrid Model for Safety Pharmacology on an Automated Patch Clamp Platform: Using Dynamic Clamp to Join iPSC-Derived Cardiomyocytes and Simulations of IK1 Ion Channels in Real-Time
Patchliner Publication in Frontiers in Physiology Authors: Goversen B., Becker., N., Stölzle-Feix S., Obergrussberger A., Vos M.A., van Veen T.A.B., Fertig N., de Boer T.P.

An important aspect of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) proposal is the use of human stem cell-derived cardiomyocytes and the confirmation of their predictive power in drug safety assays. The benefits of this cell source are clear; drugs can be tested in vitro on human cardiomyocytes, with patient-specific genotypes if needed, and differentiation efficiencies are generally excellent, resulting in a virtually limitless supply of cardiomyocytes. There are, however, several challenges that will have to be surmounted before successful establishment of hSC-CMs as an all-round predictive model for drug safety assays. An important factor is the relative electrophysiological immaturity of hSC-CMs, which limits arrhythmic responses to unsafe drugs that are pro-arrhythmic in humans. Potentially, immaturity may be improved functionally by creation of hybrid models, in which the dynamic clamp technique joins simulations of lacking cardiac ion channels (e.g., IK1) with hSC-CMs in real-time during patch clamp experiments. This approach has been used successfully in manual patch clamp experiments, but throughput is low. In this study, we combined dynamic clamp with automated patch clamp of iPSC-CMs in current clamp mode, and demonstrate that IK1 conductance can be added to iPSC-CMs on an automated patch clamp platform, resulting in an improved electrophysiological maturity.

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2017 – Myoclonus Epilepsy and Ataxia due to KCNC1 Mutation: Analysis of 20 Cases and K+ Channel Properties
Patchliner Publication in Annals in Neurobiology (2017) Authors: Oliver K.L., Franceschetti S., Milligan C.J., Muona M., Mandelstam S.A., Canafoglia L., Boguszewska-Chachulska A.M., Korczyn A.D.,Bisulli F., Di BoNaVentura C., Ragona F., Michelucci R., Ben-Zeev B., Straussberg R., Panzica F., Massano J., Friedman D., Crespel A., Engelsen B.A., Andermann F., Andermann E., Spodar K., Lasek-Bal A., Riguzzi P.,Pasini E., Tinuper P., Licchetta L., Gardella E., Lindenau M., Wulf A., Møller R.S.

Objective:To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever.Methods:We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV3.1 channels.Results:Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic–clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG–electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV3.1, increasing channel availability.Interpretation:MEAK has a relatively homogeneous presentation, resembling Unverricht–Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV3.1 subunit–containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics.

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2017 – Purinergic receptors P2RX4 and P2RX7 in familial multiple sclerosis
Patchliner Publication in Human Mutation (2017) Authors: Sadovnick A.D., Gu B.J., Traboulsee A.L., Bernales C.Q., Encarnacion M., Yee I.M., Criscuoli M.G., Huang X., Ou A., Milligan C.J., Petrou S, Wiley J.S, Vilariño-Güell C.

Genetic variants in the purinergic receptors P2RX4 and P2RX7 have been shown to affect susceptibility to multiple sclerosis (MS). In this study, we set out to evaluate whether rare coding variants of major effect could also be identified in these purinergic receptors. Sequencing analysis of P2RX4 and P2RX7 in 193 MS patients and 100 controls led to the identification of a rare three variant haplotype (P2RX7 rs140915863:C>T [p.T205M], P2RX7 rs201921967:A>G [p.N361S], and P2RX4 rs765866317:G>A [p.G135S]) segregating with disease in a multi-incident family with six family members diagnosed with MS (logarithm of odds = 3.07). Functional analysis of this haplotype in HEK293 cells revealed impaired P2X7 surface expression (P  0.01), resulting in over 95% inhibition of adenosine triphosphate (ATP)-induced pore function (P  0.001) and a marked reduction in phagocytic ability (P  0.05). In addition, transfected cells showed 40% increased peak ATP-induced inward current (P  0.01), and a greater Ca2+ response to the P2X4 135S variant compared with wild type (P  0.0001). Our study nominates rare genetic variants in P2RX4 and P2RX7 as major genetic contributors to disease, further supporting a role for these purinergic receptors in MS and the disruption of transmembrane cation channels leading to impairment of phagocytosis as the pathological mechanisms of disease.

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2017 – Identification of Na+/K+-ATPase inhibition-independent proarrhythmic ionic mechanisms of cardiac glycosides
Patchliner Publication in Nature Scientific Reports (2017) Authors: Koh C.H., Wu J., Chung Y.Y., Liu Z., Zhang R.R., Chong K., Korzh V., Ting S., Oh S., Shim W., Tian H.Y., Wei H.

The current study explored the Na+/K+-ATPase (NKA) inhibition-independent proarrhythmic mechanisms of cardiac glycosides (CGs) which are well-known NKA inhibitors. With the cytosolic Ca2+ chelated by EGTA and BAPTA or extracellular Ca2+ replaced by Ba2+, effects of bufadienolides (bufalin (BF) and cinobufagin (CBG)) and cardenolides (ouabain (Oua) and pecilocerin A (PEA)) on the L-type calcium current (I Ca,L) were recorded in heterologous expression CaV1.2-CHO cells and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). BF and CBG demonstrated a concentration-dependent (0.1 to100 µM) I Ca,L inhibition (maximal ≥50%) without and with the NKA activity blocked by 10 µM Oua. BF significantly shortened the action potential duration at 1.0 µM and shortened the extracellular field potential duration at 0.01~1.0 µM. On the other hand, BF and CBG at 100 µM demonstrated a strong inhibition (≥40%) of the rapidly activating component of the delayed rectifier K+ current (I Kr) in heterologous expression HEK293 cells and prolonged the APD of the heart of day-3 Zebrafish larva with disrupted rhythmic contractions. Moreover, hESC-CMs treated with BF (10 nM) for 24 hours showed moderate yet significant prolongation in APD90. In conclusion, our data indicate that CGs particularly bufadienolides possess cytosolic [Ca2+]i- and NKA inhibition- independent proarrhythmic potential through I Ca,L and I Kr inhibitions.

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2017 – L-Type Calcium Channel Inhibition Contributes to the Proarrhythmic Effects of Aconitine in Human Cardiomyocytes
Patchliner Publication in PLoS ONE (2017) Authors: Wu J., Wang X., Chung Y.Y. Koh C.H. Liu Z., Guo H., Yuan Q., Wang C., Su S., Wei H.

Aconitine (ACO) is well-known for causing lethal ventricular tachyarrhythmias. While cardiac Na+ channel opening during repolarization has long been documented in animal cardiac myocytes, the cellular effects and mechanism of ACO in human remain unexplored. This study aimed to assess the proarrhythmic effects of ACO in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ACO concentration-dependently (0.3 ~ 3.0 μM) shortened the action potentials (AP) durations (APD) in ventricular-like hiPSC-CMs by > 40% and induced delayed after-depolarization. Laser-scanning confocal calcium imaging analysis showed that ACO decreased the duration and amplitude of [Ca2+]i transients and increased in the beating frequencies by over 60%. Moreover, ACO was found to markedly reduce the L-type calcium channel (LTCC) currents (ICa,L) in hiPSC-CMs associated with a positive-shift of activation and a negative shift of inactivation. ACO failed to alter the peak and late Na+ currents (INa) in hiPSC-CMs while it drastically increased the late INa in Guinea-pig ventricular myocytes associated with enhanced activation/delayed inactivation of INa at -55 mV~ -85 mV. Further, the effects of ACO on ICa,L, INa and the rapid delayed rectifier potassium current (IKr) were validated in heterologous expression systems by automated voltage-clamping assays and a moderate suppression of IKr was observed in addition to concentration-dependent ICa,L inhibition. Lastly, increased beating frequency, decreased Ca2+ wave and shortened field potential duration were recorded from hiPSC-CMs by microelectrode arrays assay. In summary, our data demonstrated that LTCC inhibition could play a main role in the proarrhythmic action of ACO in human cardiomyocytes.

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2017 – Discovery of benzimidazole derivatives as modulators of mitochondrial function: A potential treatment for Alzheimer’s disease
Patchliner Publication in PLoS ONE (2017) Authors: Kim T., Yang H.Y., Park B.G., Jung S.Y., Park J.H., Park K.D., Min S.J., Tae J., Yang H., Cho S., Cho S.J., Song H., Mook-Jung I., Lee J., Pae A.N.

In this study, we designed a library of compounds based on the structures of well-known ligands of the 18 kDa translocator protein (TSPO), one of the putative components of the mPTP. We performed diverse mitochondrial functional assays to assess their ability to restore cells from Aβ-induced toxicity in vitro and in vivo. Among tested compounds, compound 25 effectively improved cognitive function in animal models of AD. Given the excellent in vitro and in vivo activity and a favorable pharmacokinetic profile of compound 25, we believe that it can serve as a promising lead compound for a potential treatment option for AD.

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2017 – ‘Gardos Channelopathy’: a variant of hereditary Stomatocytosis with complex molecular regulation
Patchliner Publication in Scientific Reports (2017) Authors: Fermo E., Bogdanova A., etkova-Kirova P., Zaninoni A., Marcello A.P., Makhro A., Hänggi P., Hertz L., Danielczok J., Vercellati C., Mirra N., Zanella A., Cortelezzi A., Barcellini W., Kaestner L., Bianchi P.

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.

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2017 – Correlation between human ether-a-go-go related gene channel inhibition and action potential prolongation
Patchliner Publication in British Journal of Pharmacology (2017) Authors: Saxena P., Hortigon‐Vinagre M.P., Beyl S.,Baburin I., Andranovits S., Iqbal S.M., Costa A., IJzerman A.P., Kügler P., Timin E., Smith G.L., Hering S.

BACKGROUND AND PURPOSE:Human ether-a-go-go-related gene (hERG; KV 11.1) channel inhibition is a widely accepted predictor of cardiac arrhythmia. hERG channel inhibition alone is often insufficient to predict pro-arrhythmic drug effects. This study used a library of dofetilide derivatives to investigate the relationship between standard measures of hERG current block in an expression system and changes in action potential duration (APD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The interference from accompanying block of CaV1.2 and NaV1.5 channels was investigated along with an in silico AP model.EXPERIMENTAL APPROACH:Drug-induced changes in APD were assessed in hiPSC-CMs using voltage-sensitive dyes. The IC50 values for dofetilide and 13 derivatives on hERG current were estimated in an HEK293 expression system. The relative potency of each drug on APD was estimated by calculating the dose (D150 ) required to prolong the APD at 90% (APD90 ) repolarization by 50%.KEY RESULTS:The D150 in hiPSC-CMs was linearly correlated with IC50 of hERG current. In silico simulations supported this finding. Three derivatives inhibited hERG without prolonging APD, and these compounds also inhibited CaV1.2 and/or NaV1.5 in a channel state-dependent manner. Adding CaV 1.2 and NaV 1.2 block to the in silico model recapitulated the direction but not the extent of the APD change.CONCLUSIONS AND IMPLICATIONS:Potency of hERG current inhibition correlates linearly with an index of APD in hiPSC-CMs. The compounds that do not correlate have additional effects including concomitant block of CaV1.2 and/or NaV1.5 channels. In silico simulations of hiPSC-CMs APs confirm the principle of the multiple ion channel effects.

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2017 – Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function
Patchliner Publication in Frontiers in Pharmacology (2017) Authors: Billet A.,Froux L., Hanrahan J.W., Becq F.

The chloride (Cl-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl- currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

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2016 – The potassium channels TASK2 and TREK1 regulate functional differentiation of murine skeletal muscle cells
Patchliner Publication in American Journal of Physiology - Cell Physiology (2016) Authors: Afzali A.M., Ruck T., Herrmann A.M., Iking J., Sommer C., Kleinschnitz C., Preuβe C., Stenzel W., Budde T., Wiendl H., Bittner S., Meuth S.G.

Two-pore domain potassium (K2P) channels influence basic cellular parameters such as resting membrane potential, cellular excitability, or intracellular Ca2+-concentration [Ca2+]i While the physiological importance of K2P channels in different organ systems (e.g., heart, central nervous system, or immune system) has become increasingly clear over the last decade, their expression profile and functional role in skeletal muscle cells (SkMC) remain largely unknown. The mouse SkMC cell line C2C12, wild-type mouse muscle tissue, and primary mouse muscle cells (PMMs) were analyzed using quantitative PCR, Western blotting, and immunohistochemical stainings as well as functional analysis including patch-clamp measurements and Ca2+ imaging. Mouse SkMC express TWIK-related acid-sensitive K+ channel (TASK) 2, TWIK-related K+ channel (TREK) 1, TREK2, and TWIK-related arachidonic acid stimulated K+ channel (TRAAK). Except TASK2 all mentioned channels were upregulated in vitro during differentiation from myoblasts to myotubes. TASK2 and TREK1 were also functionally expressed and upregulated in PMMs isolated from mouse muscle tissue. Inhibition of TASK2 and TREK1 during differentiation revealed a morphological impairment of myoblast fusion accompanied by a downregulation of maturation markers. TASK2 and TREK1 blockade led to a decreased K+ outward current and a decrease of ACh-dependent Ca2+ influx in C2C12 cells as potential underlying mechanisms. K2P-channel expression was also detected in human muscle tissue by immunohistochemistry pointing towards possible relevance for human muscle cell maturation and function. In conclusion, our findings for the first time demonstrate the functional expression of TASK2 and TREK1 in muscle cells with implications for differentiation processes warranting further investigations in physiologic and pathophysiologic scenarios.

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2017 – 14-3-3 proteins regulate K2P5.1 surface expression on T lymphocytes
Patchliner Publication in Traffic (2017) Authors: Fernández-Orth J., Ehling P., Ruck T., Pankratz S., Hofmann M.,Landgraf P., Dieterich D.C., SmallaK.-H., Kähne T., Seebohm G., Budde T., Wiendl H., Bittner S., Meuth S.G.

K2P5.1 channels (also called TASK-2 or Kcnk5) have already been shown to be relevant in the pathophysiology of autoimmune disease because they are known to be upregulated on peripheral and central T lymphocytes of multiple sclerosis (MS) patients. Moreover, overexpression of K2P5.1 channels in vitro provokes enhanced T-cell effector functions. However, the molecular mechanisms regulating intracellular K2P5.1 channel trafficking are unknown so far. Thus, the aim of the study is to elucidate the trafficking of K2P5.1 channels on T lymphocytes. Using mass spectrometry analysis, we have identified 14-3-3 proteins as novel binding partners of K2P5.1 channels. We show that a non-classical 14-3-3 consensus motif (R-X-X-pT/S-x) at the channel's C-terminus allows the binding between K2P5.1 and 14-3-3. The mutant K2P5.1/S266A diminishes the protein-protein interaction and reduces the amplitude of membrane currents. Application of a non-peptidic 14-3-3 inhibitor (BV02) significantly reduces the number of wild-type channels in the plasma membrane, whereas the drug has no effect on the trafficking of the mutated channel. Furthermore, blocker application reduces T-cell effector functions. Taken together, we demonstrate that 14-3-3 interacts with K2P5.1 and plays an important role in channel trafficking.

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2016 – Myrsinane, Premyrsinane, and Cyclomyrsinane Diterpenes fromEuphorbia falcata as Potassium Ion Channel Inhibitors with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effects
Patchliner Publication in Journal of Natural Products (2016) Authors: Vasas A., Forgo P., Orvos P., Tálosi L., Csorba A., Pinke G., Hohmann J.

GIRK channels are activated by a large number of G protein-coupled receptors and regulate the electrical activity of neurons, cardiac atrial myocytes, and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the pathophysiology of neuropathic pain, drug addiction, and cardiac arrhythmias. In the heart, GIRK channels are selectively expressed in the atrium, and their activation inhibits pacemaker activity, thereby slowing the heart rate. In the present study, 19 new diterpenes, falcatins A–S, and the known euphorprolitherin D were isolated from Euphorbia falcata. The compounds were assayed on stable transfected HEK-hERG (KV11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cells. Blocking activity on GIRK channels was exerted by 13 compounds (61–83% at 10 μM), and, among them, five possessed low potency on the hERG channel (4–20% at 10 μM). These selective activities suggest that myrsinane-related diterpenes are potential lead compounds for the treatment of atrial fibrillation.

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2016 – The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts
Patchliner Publication in Epilepsia (2016) Authors: Petty S.J., Milligan C.J., Todaro M., Richards K.L., Kularathna P.K., Pagel C.N., French C.R., Hill-Yardin E.L., O'Brien T.J., Wark J.D., Mackie E.J., Petrou S.

Objective:Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels.Methods:Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV. Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm).Results:NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p 0.001).Significance:Mouse osteoblasts express NaV, and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.

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2016 – Human EAG channels are directly modulated by PIP2 as revealed by electrophysiological and optical interference investigations
Patchliner Publication in Scientific Reports (2016) Authors: Han B., He K., Cai C., Tang Y., Yang L., Heinemann S.H., Hoshi T., Hou S.

Voltage-gated ether à go-go (EAG) K+ channels are expressed in various types of cancer cells and also in the central nervous system. Aberrant overactivation of human EAG1 (hEAG1) channels is associated with cancer and neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes. Although hEAG1 channels are recognized as potential therapeutic targets, regulation of their functional properties is only poorly understood. Here, we show that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent inhibitory gating modifier of hEAG1 channels. PIP2 inhibits the channel activity by directly binding to a short N-terminal segment of the channel important for Ca2+/calmodulin (CaM) binding as evidenced by bio-layer interferometry measurements. Conversely, depletion of endogenous PIP2 either by serotonin-induced phospholipase C (PLC) activation or by a rapamycin-induced translocation system enhances the channel activity at physiological membrane potentials, suggesting that PIP2 exerts a tonic inhibitory influence. Our study, combining electrophysiological and direct binding assays, demonstrates that hEAG1 channels are subject to potent inhibitory modulation by multiple phospholipids and suggests that manipulations of the PIP2 signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases.

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2016 – Human T cells in silico: Modelling their electrophysiological behaviour in health and disease
Patchliner Publication in Journal of Theoretical Biology (2016) Authors: Ehling P., Meuth P., Eichinger P., Hermann A.M., Bittner S., Pawlowski M., Pankratz S., Herty M., Budde T., Meuth S.G.

Although various types of ion channels are known to have an impact on human T cell effector functions, their exact mechanisms of influence are still poorly understood. The patch clamp technique is a well-established method for the investigation of ion channels in neurons and T cells. However, small cell sizes and limited selectivity of pharmacological blockers restrict the value of this experimental approach. Building a realistic T cell computer model therefore can help to overcome these kinds of limitations as well as reduce the overall experimental effort. The computer model introduced here was fed off ion channel parameters from literature and new experimental data. It is capable of simulating the electrophysiological behaviour of resting and activated human CD4+ T cells under basal conditions and during extracellular acidification. The latter allows for the very first time to assess the electrophysiological consequences of tissue acidosis accompanying most forms of inflammation.

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2015 – The two-pore domain K2P channel TASK2 drives human NK-cell proliferation and cytolytic function
Patchliner Publication in European Journal of Immunology (2015) Authors: Schulte-Mecklenbeck A., Bittner S., Ehling P., Döring F., Wischmeyer E., Breuer J., Herrmann A.M., Wiendl H., Meuth S.G., Gross C.C.

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that recognize and kill tumor‐ and virus‐infected cells without prior stimulation. Killing of target cells is a multistep process including adhesion to target cells, formation of an immunological synapse, and polarization and release of cytolytic granules. The role of distinct potassium channels in this orchestrated process is still poorly understood. The current study reveals that in addition to the voltage‐gated KV1.3 and the calcium‐activated KCa3.1 channels, human NK cells also express the two‐pore domain K2P channel TASK2 (TWIK‐related acid‐sensitive potassium channel). Expression of Task2 varies among NK‐cell subsets and depends on their differentiation and activation state. Despite its different expression in TASK2highCD56brightCD16− and TASK2lowCD56dimCD16+ NK cells, TASK2 is involved in cytokine‐induced proliferation and cytolytic function of both subsets. TASK2 is crucial for leukocyte functional antigen (LFA‐1) mediated adhesion of both resting and cytokine‐activated NK cells to target cells, an early step in killing of target cells. With regard to the following mechanism, TASK2 plays a role in release of cytotoxic granules by resting, but not IL‐15‐induced NK cells. Taken together, our data exhibit two‐pore potassium channels as important players in NK‐cell activation and effector function.

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2016 – Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes
Patchliner Publication in Stem Cells and Development (2016) Authors: Rajamohan D., Kalra S., Hoang M.D., George V., Staniforth A., Russell H., Yang X., Denning C.

Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose–response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation.

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2014 – Physiological and genetic analysis of multiple sodium channel variants in a model of genetic absence epilepsy
Patchliner Publication in Neurobiology of Disease (2014) Authors: Oliva M.K., McGarr T.C., Beyer B.J., Gazina E., Kaplan D.I., Cordeiro L., Thomas E., Dib-Hajj S.D., Waxman S.G., Frankel W.N., Petrou S.

In excitatory neurons, SCN2A (NaV1.2) and SCN8A (NaV1.6) sodium channels are enriched at the axon initial segment. NaV1.6 is implicated in several mouse models of absence epilepsy, including a missense mutation identified in a chemical mutagenesis screen (Scn8aV929F). Here, we confirmed the prior suggestion that Scn8aV929F exhibits a striking genetic background-dependent difference in phenotypic severity, observing that spike-wave discharge (SWD) incidence and severity are significantly diminished when Scn8aV929F is fully placed onto the C57BL/6J strain compared with C3H. Examination of sequence differences in NaV subunits between these two inbred strains suggested NaV1.2V752F as a potential source of this modifier effect. Recognising that the spatial co-localisation of the NaV channels at the axon initial segment (AIS) provides a plausible mechanism for functional interaction, we tested this idea by undertaking biophysical characterisation of the variant NaV channels and by computer modelling. NaV1.2V752F functional analysis revealed an overall gain-of-function and for NaV1.6V929F revealed an overall loss-of-function. A biophysically realistic computer model was used to test the idea that interaction between these variant channels at the AIS contributes to the strain background effect. Surprisingly this modelling showed that neuronal excitability is dominated by the properties of NaV1.2V752F due to “functional silencing” of NaV1.6V929F suggesting that these variants do not directly interact. Consequent genetic mapping of the major strain modifier to Chr 7, and not Chr 2 where Scn2a maps, supported this biophysical prediction. While a NaV1.6V929F loss of function clearly underlies absence seizures in this mouse model, the strain background effect is apparently not due to an otherwise tempting Scn2a variant, highlighting the value of combining physiology and genetics to inform and direct each other when interrogating genetic complex traits such as absence epilepsy.

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2015 – Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy
Patchliner Publication in Journal of Clinical Medicine (2015) Authors: Friedrichs S., Malan D., Voss Y., Sasse P.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening

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2013 – Red cell investigations: Art and artefacts
Patchliner Publication in Blood reviews (2013) Authors: Minetti G., Egée S., Mörsdorf D., Steffen P., Makhro A., Achilli C., Ciana A., Wang J., Bouyer G., Bernhardt I., Wagner C., Thomas S., Bogdanova A., Kaestner L.

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

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2014 – Multi-Generational Pharmacophore Modeling for Ligands to the Cholane Steroid-Recognition Site in the β1 Modulatory Subunit of the BK(Ca) Channel
Patchliner Publication in Journal of Molecular Graphics and Modelling (2014) Authors: McMillan J.E., Bukiya A.N., Terrell C.L., Patil S.A., Miller D.D., Dopico A.M., Parrilla A.L.

Large conductance, voltage- and Ca2+-gated K+ (BKCa) channels play a critical role in smooth muscle contractility and thus represent an emerging therapeutic target for drug development to treat vascular disease, gastrointestinal, bladder and uterine disorders. Several compounds are known to target the ubiquitously expressed BKCa channel-forming α subunit. In contrast, just a few are known to target the BKCa modulatory β1 subunit, which is highly expressed in smooth muscle and scarce in most other tissues. Lack of available high-resolution structural data makes structure-based pharmacophore modeling of β1 subunit-dependent BKCa channel activators a major challenge. Following recent discoveries of novel BKCa channel activators that act via β1 subunit recognition, we performed ligand-based pharmacophore modeling that led to the successful creation and fine-tuning of a pharmacophore over several generations. Initial models were developed using physiologically active cholane steroids (bile acids) as template. However, as more compounds that act on BKCa β1 have been discovered, our model has been refined to improve accuracy. Database searching with our best-performing model has uncovered several novel compounds as candidate BKCa β1 subunit ligands. Eight of the identified compounds were experimentally screened and two proved to be activators of recombinant BKCa β1 complexes. One of these activators, sobetirome, differs substantially in structure from any previously reported activator.

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2013 – Establishment of a Secondary Screening Assay for P/Q-Type Calcium Channel Blockers
Patchliner Publication in Combinatorial Chemistry & High Throughput Screening (2013) Authors: Hermann D., Mezler M., Swensen A., Bruehl C., Obergrußberger A., Wicke K., Schoemaker H., Gross G., Draguhn A., Nimmrich V.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels.We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steadystate inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings.Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and – if necessary – a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs

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2013 – Minimized cell usage for stem cell-derived and primary cells on an automated patch clamp system
Patchliner Publication in Journal of Pharmacological and Toxicological Methods (2013) Authors: Becker N., Stoelzle S., Göpel S., Guinot D., Mumm P., Haarmann C., Malan D., Bohlen H., Kossolov E., Kettenhofen R., George M., Fertig N., Brüggemann A.

IntroductionChip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons.MethodsWe established application protocols for CHO cells, IPS cell-derived neurons (iCell® Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U®, Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 μl cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture.ResultsWe present a new cell application procedure optimized for the Patchliner achieving > 80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application.DiscussionOur new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability.

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2012 – TRPA1 Agonist Activity of Probenecid Desensitizes Channel Responses: Consequences for Screening
Patchliner Publication in ASSAY and Drug Development Technologies (2012) Authors: McClenaghan C., Zeng F., Verkuyl J.M.

The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 μM (P0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.

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2013 – Automated Planar Patch Clamp
Patchliner book chapter in Ion Channels (2013) Authors: Milligan, C. J., Möller, C.

Ion channels are integral membrane proteins that regulate the  flow of ions across the plasma membrane and the membranes of intracellular organelles of both excitable and non-excitable cells. Ion channels are vital to a wide variety of biological processes and are prominent components of the nervous system and cardiovascular system, as well as controlling many metabolic functions. Furthermore, ion channels are known to be involved in many disease states and as such have become popular therapeutic targets. For many years now manual patch-clamping has been regarded as one of the best approaches for assaying ion channel function, through direct measurement of ion  flow across these membrane proteins. Over the last decade there have been many remarkable breakthroughs in the development of technologies enabling the study of ion channels. One of these breakthroughs is the development of automated planar patch-clamp technology. Automated platforms have demonstrated the ability to generate high-quality data with high throughput capabilities, at great efficiency and reliability. Additional features such as simultaneous intracellular and extracellular perfusion of the cell membrane, current clamp operation, fast compound application, an increasing rate of parallelization, and more recently temperature control have been introduced. Furthermore, in addition to the well-established studies of over-expressed ion channel proteins in cell lines, new generations of planar patch-clamp systems have enabled successful studies of native and primary mammalian cells. This technology is becoming increasingly popular and extensively used both within areas of drug discovery as well as academic research. Many platforms have been developed including NPC-16 Patchliner and SyncroPatch 96 (Nanion Technologies GmbH, Munich), CytoPatch™ (Cytocentrics AG, Rostock), PatchXpress ®  7000A, IonWorks ®  Quattro and IonWorks Barracuda™, (Molecular Devices, LLC); Dyna flow ®  HT (Cellectricon AB, Mölndal), QPatch HT (Sophion A/S, Copenhagen), IonFlux HT (Fluxion Bioscience Inc, USA), which have demonstrated the capability to generate recordings similar in quality to that of conventional patch clamping. Here we describe features of Nanion’s NPC-16 Patchliner and processes and protocols suited for this particularly flexible and successful high-throughput automated platform, which is based on planar patch-clamp technology. However, many of the protocols and notes given in this chapter can be applied to other automated patch-clamp platforms, similarly.

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2012 – Synthesis and In Vitro Antibacterial Activity of Novel 3‐Azabicyclo [3.3. 0] octanyl Oxazolidinones
Patchliner Publication in Chemical Biology and Drug Design (2012) Authors: Bhattarai D., Lee S.H., Seo S.H., Nam G., Kang S.B., Pae A.N., Kim E.E., Oh T., Cho S.N., Keum G.

We synthesized a series of oxazolidinone-type antibacterials in which morpholine C-ring of linezolid has been modified by substituted 3-azabicyclo[3.3.0]octanyl rings. Acetamide or 1,2,3-triazole heterocycle was used as C-5 side chain of oxazolidinone. The resulting series of compounds was then screened in vitro against panel of susceptible and resistant Gram-positive, Gram-negative bacteria, and Mycobacterium tuberculosis (Mtb). Several analogs in this series exhibited potent in vitro antibacterial activity comparable or superior to linezolid against the tested bacteria. Compounds 10a, 10b, 11a, and 15a displayed highly potent activity against M. tuberculosis. Selected compound 10b showed good human microsomal stability and CYP-profile, and showed low activity against hERG channel.

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2012 – Toward a new gold standard for early safety: automated temperature-controlled hERG test on the Patchliner
Patchliner Publication in Frontiers in Pharmacology (2012) Authors: Polonchuk L.

The Patchliner temperature-controlled automated patch clamp system was evaluated for testing drug effects on potassium currents through human ether-à-go-go related gene (hERG) channels expressed in Chinese hamster ovary cells at 35–37°C. IC50 values for a set of reference drugs were compared with those obtained using the conventional voltage clamp technique. The results showed good correlation between the data obtained using automated and conventional electrophysiology. Based on these results, the Patchliner represents an innovative automated electrophysiology platform for conducting the hERG assay that substantially increases throughput and has the advantage of operating at physiological temperature. It allows fast, accurate, and direct assessment of channel function to identify potential proarrhythmic side effects and sets a new standard in ion channel research for drug safety testing.

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2012 – Characterizing Human Ion Channels in Induced Pluripotent Stem Cell-Derived Neurons
Patchliner Publication in Journal of Biomolecular Screening (2012) Authors: Haythornthwaite A, Stoelzle S, Hasler A, Kiss A, Mosbacher J, George M, Brüggemann A, Fertig N.

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na+ (NaV), Ca2+ (CaV), and K+ (KV) channels as expected from excitable cells. The NaV current was TTX sensitive, IC50 = 12 ± 6 nM (n = 5). About 50% of the CaV current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the KV channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC50 = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABAA receptor antagonist bicuculline, IC50 = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed NaV currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching “excitable cells.” These results show high potential for pluripotent stem cell–derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

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2012 – Observation of Antinociceptive Effects of Oxymatrine and its Effect on Delayed Rectifier K+ Currents (Ik) in PC12 Cells
Patchliner Publication in Neurochemical Research (2012) Authors: Wang Y., Yuan J., Yuan X., Wang W., Pei X., Zhao Q., Cao H., Xu M., Liu Z.

In order to observe antinociceptive effect of Oxymatrine (OMT) and its effect on voltage-activated K+ channel, the acetic acid-induced abdominal contraction model of mouse was used to test the antinociceptive effect in vivo, and in vitro, the delayed rectifier K+ currents (Ik) in PC12 cells (rat pheochromocytoma cells) was recorded using the automated patch-clamp method. The results indicated that after application of OMT, the number of acetic acid-induced animal abdominal contraction was significantly decreased, Ik in PC12 cells was significantly decreased, and showed a concentration-dependent manner. After application of OMT, both the activation and inactivation curves of Ik of PC12 cells were shifted to negative potentials. This study revealed that OMT showed antinociceptive effect in mice. The inhibition of voltage-activated K+ channel might be one of mechanisms in which the enhanced both activation and inactivation of K+ channel were involved and might play important roles.

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2011 – Rapid and Contrasting Effects of Rosiglitazone on Transient Receptor Potential TRPM3 and TRPC5 Channels
Patchliner Publication in Molecular Pharmacology (2011) Authors: Majeed Y., Bahnasi Y., Seymour V.A.L., Wilson L.A., Milligan C.J., Agarwal A.K., Sukumar P., Naylor J., Beech D.J.

The aim of this study was to generate new insight into chemical regulation of transient receptor potential (TRP) channels with relevance to glucose homeostasis and the metabolic syndrome. Human TRP melastatin 2 (TRPM2), TRPM3, and TRP canonical 5 (TRPC5) were conditionally overexpressed in human embryonic kidney 293 cells and studied by using calcium-measurement and patch-clamp techniques. Rosiglitazone and other peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists were investigated. TRPM2 was unaffected by rosiglitazone at concentrations up to 10 μM but was inhibited completely at higher concentrations (IC50, ∼22.5 μM). TRPM3 was more potently inhibited, with effects occurring in a biphasic concentration-dependent manner such that there was approximately 20% inhibition at low concentrations (0.1–1 μM) and full inhibition at higher concentrations (IC50, 5–10 μM). PPAR-γ antagonism by 2-chloro-5-nitrobenzanilide (GW9662) did not prevent inhibition of TRPM3 by rosiglitazone. TRPC5 was strongly stimulated by rosiglitazone at concentrations of ≥10 μM (EC50, ∼30 μM). Effects on TRPM3 and TRPC5 occurred rapidly and reversibly. Troglitazone and pioglitazone inhibited TRPM3 (IC50, 12 μM) but lacked effect on TRPC5, suggesting no relevance of PPAR-γ or the thiazolidinedione moiety to rosiglitazone stimulation of TRPC5. A rosiglitazone-related but nonthiazolidinedione PPAR-γ agonist, N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-l-tyrosine (GW1929), was a weak stimulator of TRPM3 and TRPC5. The natural PPAR-γ agonist 15-deoxy prostaglandin J2, had no effect on TRPM3 or TRPC5. The data suggest that rosiglitazone contains chemical moieties that rapidly, strongly, and differentially modulate TRP channels independently of PPAR-γ, potentially contributing to biological consequences of the agent and providing the basis for novel TRP channel pharmacology.

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2011 – The Chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation
Patchliner Publication in Journal of Biological Chemistry (2011) Authors: Papakosta M., Dalle C., Haythornthwaite A., Cao L., Stevens E.B., Burgess G., Russell R., Cox P.J., Phillips S.C., Grimm C.

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

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2011 – Development of a selective small-molecule inhibitor of Kir1.1, the Renal Outer Medullary Potassium Channel
Patchliner Publication in Molecular Pharmacology (2011) Authors: Bhave G., Chauder B.A., Liu W., Dawson E.S., Kadakia R., Nguyen T.T., Lewis L.M., Meiler J., Weaver C.D., Satlin L.M., Lindsley C.W., Denton J.S.

The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2′-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target.

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2011 – Pharmacological studies of CaV3.1 T-type calcium channels using automated patch-clamp techniques
Patchliner Publication in General Physiology and Biophysics (2011) Authors: Choi K.-H., Song C., Cheong C.S., Rhim H.

T-type calcium channels are involved in a variety of physiological and pathophysiological processes, and thus could be therapeutic targets. However, there is no T-type channel selective blocker for use in clinical practice, demanding a need for the development of novel drugs where a higher-throughput screening system is required. Here we present pharmacological studies on CaV3.1 T-type channels using automated patch-clamp. The IC50 values obtained from automated patch-clamp and conventional one showed a good correlation (correlation coefficient of 0.82), suggesting that the automated patch-clamp is an efficient and reliable method for ranking the drug potencies for T-type channels.

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2010 – Pregnenolone Sulphate- and Cholesterol-Regulated TRPM3 Channels Coupled to Vascular Smooth Muscle Secretion and Contraction
Patchliner Publication in Circulation Research (2010) Authors: Naylor J., Li J., Milligan C.J, Zeng F., Sukumar P., Hou B., Sedo A., Yuldasheva N., Majeed Y., Beri D., Jiang S., Seymour V.A.L, McKeown L., Kumar B., Harteneck C., O'Regan D., Wheatcroft S.B., Kearney M.T, Jones C., Porter K.E., Beech D.J.

Rationale: Transient receptor potential melastatin (TRPM)3 is a calcium-permeable ion channel activated by the neurosteroid pregnenolone sulfate and positively coupled to insulin secretion in β cells. Although vascular TRPM3 mRNA has been reported, there is no knowledge of TRPM3 protein or its regulation and function in the cardiovascular system. Objective: To determine the relevance and regulation of TRPM3 in vascular biology. Methods and Results: TRPM3 expression was detected at mRNA and protein levels in contractile and proliferating vascular smooth muscle cells. Calcium entry evoked by pregnenolone sulfate or sphingosine was suppressed by TRPM3 blocking antibody or knock-down of TRPM3 by RNA interference. Low-level constitutive TRPM3 activity was also detected. In proliferating cells, channel activity was coupled negatively to interleukin-6 secretion via a calcium-dependent mechanism. In freshly isolated aorta, TRPM3 positively modulated contractile responses independently of L-type calcium channels. Concentrations of pregnenolone sulfate required to evoke responses were higher than the known plasma concentrations of the steroids, leading to a screen for other stimulators. β-Cyclodextrin was one of few stimulators of TRPM3, revealing the channels to be partially suppressed by endogenous cholesterol, the precursor of pregnenolone. Elevation of cholesterol further suppressed channel activity and loading with cholesterol to generate foam cells precluded observation of TRPM3 activity. Conclusions: The data suggest functional relevance of TRPM3 in contractile and proliferating phenotypes of vascular smooth muscle cells, significance of constitutive channel activity, regulation by cholesterol, and potential value of pregnenolone sulfate in therapeutic vascular modulation.

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2010 – Propranolol blocks cardiac and neuronal voltage-gated sodium channels
Patchliner Publication in Frontiers in Pharmacology (2010) Authors: Wang D.W., Mistry A.M., Kahlig K.M., Kearney J.A, Xiang J., George Jr. A.L.

Propranolol is a widely used, non-selective β-adrenergic receptor antagonist with proven efficacy in treating cardiovascular disorders and in the prevention of migraine headaches. At plasma concentrations exceeding those required for β-adrenergic receptor inhibition, propranolol also exhibits anti-arrhythmic (“membrane stabilizing”) effects that are not fully explained by β-blockade. Previous in vitro studies suggested that propranolol may have local anesthetic effects. We directly tested the effects of propranolol on heterologously expressed recombinant human cardiac (NaV1.5) and brain (NaV1.1, NaV1.2, NaV1.3) sodium channels using whole-cell patch-clamp recording. We found that block was not stereospecific as we observed approximately equal IC50 values for tonic and use-dependent block by R-(+) and S-(−) propranolol (tonic block: R: 21.4 μM vs S: 23.6 μM; use-dependent block: R: 2.7 μM vs S: 2.6 μM). Metoprolol and nadolol did not block NaV1.5 indicating that sodium channel block is not a class effect of β-blockers. The biophysical effects of R-(+)-propranolol on NaV1.5 and NaV1.1 resembled that of the prototypical local anesthetic lidocaine including the requirement for a critical phenylalanine residue (F1760 in NaV1.5) in the domain 4 S6 segment. Finally, we observed that brain sodium channels exhibited less sensitivity to R-(+)-propranolol than NaV1.5 channels. Our findings establish sodium channels as targets for propranolol and may help explain some beneficial effects of the drug in treating cardiac arrhythmias, and may explain certain adverse central nervous system effects.

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2010 – Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae
Patchliner Publication in Bioorganic & Medical Chemistry (2010) Authors: Balansa W, Islam R, Fontaine F, Piggott AM, Zhang H, Webb TI, Gilbert DF, Lynch JW, Capon RJ.

Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure–activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against α1 and α3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.

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2010 – Multiplexed transposon-mediated stable gene transfer in human cells
Patchliner Publication in Proc. Natl. Acad. Sci. USA. (2010) Authors: Kahlig K.M., Saridey S.K., Kaja A., Daniels M.A., George A.L. Jr., Wilson M.H.

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes.

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2009 – High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1
Patchliner Publication in Molecular Pharmacology (2009) Authors: Lewis L.M., Bhave G., Chauder B.A., Banerjee S., Lornsen K.A., Redha R., Fallen K., Lindsley C.W., Weaver C.D., Denton J.S.

The renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K+ (Kir) channels expressed in the nephron, such as Kir7.1, are less well understood in part due to the lack of selective pharmacological probes targeting inward rectifiers. In an effort to identify Kir channel probes, we performed a fluorescence-based, high-throughput screen (HTS) of 126,009 small molecules for modulators of ROMK function. Several antagonists were identified in the screen. One compound, termed VU590, inhibits ROMK with submicromolar affinity, but has no effect on Kir2.1 or Kir4.1. Low micromolar concentrations inhibit Kir7.1, making VU590 the first small-molecule inhibitor of Kir7.1. Structure-activity relationships of VU590 were defined using small-scale parallel synthesis. Electrophysiological analysis indicates that VU590 is an intracellular pore blocker. VU590 and other compounds identified by HTS will be instrumental in defining Kir channel structure, physiology, and therapeutic potential.

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2009 – Robotic multiwell planar patch-clamp for native and primary mammalian cells
Patchliner Publication in Nature Protocols (2009) Authors: Milligan C.J., Li J., Sukumar P., Majeed Y., Dallas M.L., English A., Emery P., Porter K.E., Smith A.M., McFadzean I., Beccano-Kelly D., Bahnasi Y., Cheong A., Naylor J., Zeng F., Liu X., Gamper N., Jiang L., Pearson H.A., Peers C., Robertson B., Beech D.J.

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4–8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1–6 h depending on the experimental design and yields 16–33 cell recordings.

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2008 – TRPC channel activation by extracellular thioredoxin
Patchliner Publication in Nature (2008) Authors: Xu S.Z., Sukumar P., Zeng F., Li J., Jairaman A., English A., Naylor J., Ciurtin C., Majeed Y., Milligan C.J., Bahnasi Y.M., Al-Shawaf E., Porter K.E., Jiang L.H., Emery P., Sivaprasadarao A., Beech D.J.

Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5–TRPC1 heteromultimeric channels by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis, an inflammatory joint disease that disables millions of people worldwide. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5–TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellularthioredoxin to cell function.

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2009 – High throughput techniques for discovering new glycine receptor modulators and their binding sites
Patchliner Publication in Frontiers in Molecular Neuroscience (2009) Authors: Gilbert D.F.,Islam R., Lynagh T., Lynch J.W., Webb T.I. Front.

The inhibitory glycine receptor (GlyR) is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific modulators. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384-well plate using nanogram quantities of plasmid DNA.

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2008 – Interactions, functions, and independence of plasma membrane STIM1 and TRPC1 in vascular smooth muscle cells
Patchliner Publication in Circulation Research (2008) Authors: Li J., Sukumar P., Milligan C.J., Kumar B., Ma Z.Y., Munsch C.M., Jiang L.H., Porter K.E., Beech D.J.

Stromal interaction molecule 1 (STIM1) is a predicted single membrane–spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane–spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.

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2008 – Production of a specific extracellular inhibitor of TRPM3 channels
Patchliner Publication in British Journal of Pharmacology (2008) Authors: Naylor J., Milligan C.J., Zeng F., Jones C., Beech D.J.

Background and purpose: Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family. Experimental approach: A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca2+ measurement. Key results: Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca2+ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response. Conclusions and implications: The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.

Webinar
27.01.2021 | Webinar: Thinking outside the cardiac box
Speaker:
Dr. Elena Dragicevic (Senior Sales and Alliance Manager; Nanion Technologies)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are attractive due to their unlimited availability and human origin, making them a promising tool for cardiac research and safety pharmacology. However, they can show an immature phenotype such as lower inward rectifier potassium current (IK1), atypical expression pattern of ion channels, divergent response to pharmacological agents and contractile behaviour compared to adult CMs. Thus, their detailed characterization and optimized recording environments are essential.

We aimed to characterize and modulate electrophysiological and contractile properties of hiPSC-CMs using automated dynamic clamp and contraction measurements on flexible substrate.

Here, we recorded iCell Cardiomyocytes2 in voltage and current clamp using a combined automated patch clamp (APC) and dynamic clamp device (Patchliner Dynamite8), and contractility recordings were made using the FLEXcyte 96. During the APC recordings simulated IK1 and seal compensation were applied to up to 8 hiPSC-CMs simultaneously, while the contractility recordings were conducted in 96-well plates. We have tested various compounds targeting cardiac ion channels and recorded their effects on action potential duration (APD), sodium, calcium and potassium currents, as well as their effect on the contraction capabilities of these cells. Additionally, different levels of static and cyclic pressure were applied to the cell monolayers with the aim to induce membrane deflection for reproducibility test of Frank-Starling mechanism and to imitate the physiological stretching experienced by CMs in the beating human heart during systolic and diastolic phases, respectively.

Seal compensation and virtual IK1 in hiPSC-CMs resulted in more stable and longer APs with low APD variability. Consequently, the dynamic clamp approach enabled reliable calcium, sodium and potassium channel pharmacology on action potentials of these cells. Culturing conditions that support contractility, i.e. flexible membrane substrates, demonstrate Ca2+ channel pharmacology equivalent to that expected from adult CMs while applied mechanical stimulation resulted in functional changes of hiPSC-CMs physiology.

Application Note PDF
Cardiac Ion Channels – “High Throughput Screening of Cardiac Ion Channels”
SyncroPatch 384PE (a predecessor model of the SyncroPatch 384), Patchliner and CardioExcyte 96 application note   

In 2013 the Cardiac Safety Research Consortium (CSRC), the Health and Environmental Sciences Institute (HESI), and the US Food and Drug Administration (FDA) proposed a new paradigm to improve assessment of the proarrythmic risk of therapeutic compounds. Until now, drug safety testing has focussed on interaction with the hERG channel and QT prolongation which can lead to potentially fatal torsades de pointes (TdP). Although this approach has been largely successful in preventing new drugs reaching the market with unexpected potential to cause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. The new paradigm, the Comprehensive In-vitro Proarrhythmia Assay (CiPA) was introduced to provide a more complete assessment of proarrythmic risk by evaluating and implementing currently available high throughput methods. An important part of this remains electrophysiological evaluation of not only hERG, but also other cardiac channels including NaV1.5, CaV1.2, KVLQT1 and Kir2.1. Additionally, new technologies, such as impedance measurements, and cells such as stem cell-derived cardiomyocytes, may provide useful tools for high throughput safety assessment. Here, we present high quality data with reliable pharmacology on hERG expressing CHO cells, NaV1.5, CaV1.2 or KVLQT1 expressed in HEK293 cells and Kir2.1 expressed in RBL cells on the SyncroPatch 384PE or Patchliner. Additionally, electrophysiological recordings on the Patchliner and Impedance measurements on the CardioExcyte 96 of stem cell-derived cardiomyocytes are shown

Application Note PDF
Cardiomyocytes – “Combining automated patch clamp, impedance and EFP of hiPSC-CMs”
CardioExcyte 96,  SyncroPatch 384PE (a predecessor model of the SyncroPatch 384) and Patchliner Application Note    Cells kindly provided by Takara-Clonetech.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are gaining interest in cardiac safety screening. Given their recapitulation of native behavior, availability, ease of use and standardized production, they are likely to provide a viable alternative to acutely isolated cardiomyocytes to assess the pro-arrhythmic potentials of drug candidates. In 2013 the Comprehensive In-vitro Proarrhythmia Assay (CiPA) was introduced to provide a more complete assessment of pro- arrythmic risk by evaluating and implementing currently available high throughput methods and evaluating the potential use of hiPSC-CMs as a model  system for cardiac safety testing. Until now, drug safety testing has focussed on interaction with the hERG channel and QT prolongation which can lead to potentially fatal torsades de pointes (TdP). Although this approach has been largely successful in preventing new drugs reaching the market with unexpected potential to cause TdP, it is also possible that potentially valuable therapeutics have failed due to this early screening. The CiPA initiative has proposed an expansion of patch clamp assessment beyond hERG to include, e.g. NaV1.5 and CaV1.2. In addition, techniques such as multi-electrode array (MEA) and impedance are being thoroughly evaluated as complementary techniques to patch clamp. Here we present data recorded using the automated patch clamp platforms, the Patchliner, SyncroPatch 96 and SyncroPatch 384PE on Cellartis® Cardiomyocytes (Takara Bio Europe Cat nr. Y10075). Recordings of NaV1.5 and CaV1.2 are shown.  Impedance and EFP recordings were also performed using the CardioExcyte 96, and the effects of verapamil and sotalol are shown.

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2023 – Stable expression of human Nav1.5 for high-throughput cardiac safety assessment
Patchliner Publication in Molecular and Cellular Toxicology (2023) Authors: Choi J., Kim R-E., Cho Y-Y., Choi J-S.

Background

Cardiac safety of new drugs is essential for public health. Nav1.5 is the cardiac sodium channel responsible for action potentials in cardiomyocytes.

Objective

For high-throughput cardiotoxicity assays in the development of antiarrhythmic drugs, we have established an electrophysiologically validated stable HEK293 cell line expressing human Nav1.5 (hNav1.5). To validate the cell line, we examined the effects of lidocaine, an antiarrhythmic agent, and compared its effects using conventional and automated patch-clamp systems.

Results

We isolated three stable cell lines originating from a single clonal cell and selected one stable cell line that produced a minimum 5 nA of hNav1.5 currents without any change in biophysical properties compared to the current from the transiently expressed hNav1.5. We further compared the pharmacological effects of lidocaine on this cell line using the conventional patch-clamp and automated patch-clamp systems. Lidocaine blocked hNav1.5 currents in a concentration- and voltage-dependent manner. The IC50 values at holding potentials of − 90 mV, near the resting membrane potential of cardiomyocytes, and − 120 mV were 18.4 ± 2.6 μM and 775.6 ± 37.1 μM, respectively. In the automated patch-clamp system, the IC50 values at holding potentials of − 90 mV and − 120 mV were 17.9 ± 2.0 μM and 578.7 ± 74.3 μM, respectively, indicating no difference between the systems. In both systems, lidocaine caused significant shifts toward hyperpolarization in the steady-state inactivation curves by ~ 20 mV and induced slower recovery from inactivation and stronger use-dependent inhibition.

Conclusion

The new HEK293 cell line stably expressing hNav1.5 channels produced a current that could be tested using both conventional and automated patch-clamp systems with similar results. This current would be strong enough to evaluate cardiac safety, thus allowing the use of the automated patch-clamp system for drug screening and functional kinetic studies to reveal the mechanism of drug action.

How can we help you?

Contact our specialist Dr. Nadine Becker (Product Manager of the Patchliner). Nadine is delighted to help you:

Nadine.Becker@nanion.de
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