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Big conductance calcium-activated (BK) channel openers can inhibit pathologically driven neural hyperactivity to control symptoms via hyperpolarizing signals to limit neural excitability. We hypothesized that BK channel openers would be neuroprotective during neuroinflammatory, autoimmune disease. The neurodegenerative disease was induced in a mouse experimental autoimmune encephalomyelitis model with translational value to detect neuroprotection in multiple sclerosis. Following the treatment with the BK channel openers, BMS-204253 and VSN16R, neuroprotection was assessed using subjective and objective clinical outcomes and by quantitating spinal nerve content. Treatment with BMS-204253 and VSN16R did not inhibit the development of relapsing autoimmunity, consistent with minimal channel expression via immune cells, nor did it change leukocyte levels in rodents or humans. However, it inhibited the accumulation of nerve loss and disability as a consequence of autoimmunity. Therefore, in addition to symptom control, BK channel openers have the potential to save nerves from excitotoxic damage and could be useful as either stand-alone neuroprotective agents or as add-ons to current disease-modifying treatments that block relapsing MS but do not have any direct neuroprotective activity.
Read more in the publication here.
Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.
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It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.
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Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.
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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.
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Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca2+-handling properties in hiPSC-CM over long-term culture (30–80 days). hiPSC-CMs > 50 days post differentiation show significantly larger ICa,L density along with an increased ICa,L-triggered Ca2+-transient. INa and IK1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed IK1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca2+-handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research.
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Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of NaV1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.
Febrile seizures (FS) are the most common form of epilepsy in children between six months and five years of age. FS is a self-limited type of fever-related seizure. However, complicated prolonged FS can lead to complex partial epilepsy. We found that among the GABAA receptor subunit (GABR) genes, most variants associated with FS are harbored in the γ2 subunit (GABRG2). Here, we characterized the effects of eight variants in the GABAA receptor γ2 subunit on receptor biogenesis and channel function. Two-thirds of the GABRG2 variants followed the expected autosomal dominant inheritance in FS and occurred as missense and nonsense variants. The remaining one-third appeared as de novo in the affected probands and occurred only as missense variants. The loss of GABAA receptor function and dominant negative effect on GABAA receptor biogenesis likely caused the FS phenotype. In general, variants in the GABRG2 result in a broad spectrum of phenotypic severity, ranging from asymptomatic, FS, genetic epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome individuals. The data presented here support the link between FS, epilepsy, and GABRG2 variants, shedding light on the relationship between the variant topological occurrence and disease severity.
N-methyl-D-aspartate receptors (NMDARs) play vital roles in normal brain functions (i.e., learning, memory, and neuronal development) and various neuropathological conditions, such as epilepsy, autism, Parkinson’s disease, Alzheimer’s disease, and traumatic brain injury. Endogenous neuroactive steroids such as 24(S)-hydroxycholesterol (24(S)-HC) have been shown to influence NMDAR activity, and positive allosteric modulators (PAMs) derived from 24(S)-hydroxycholesterol scaffold can also enhance NMDAR function. This study describes the structural determinants and mechanism of action for 24(S)-hydroxycholesterol and two novel synthetic analogs (SGE-550 and SGE-301) on NMDAR function. We also show that these agents can mitigate the altered function caused by a set of loss-of-function missense variants in NMDAR GluN subunit-encoding GRIN genes associated with neurological and neuropsychiatric disorders. We anticipate that the evaluation of novel neuroactive steroid NMDAR PAMs may catalyze the development of new treatment strategies for GRIN-related neuropsychiatric conditions.
The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.
Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit.
To identify pore domain ligands on Kv7.2 potassium ion channels, we compared wild-type (WT) and W236L mutant Kv7.2 channels in a series of assays with previously validated and novel agonist chemotypes. Positive controls were retigabine, flupirtine, and RL-81; i.e. Kv7.2 channel activators that significantly shift voltage-dependent activation to more negative potentials (ΔV50) at 5 µM. We identified 6 new compounds that exhibited differential enhancing activity between WT and W236L mutant channels. Whole cell patch-clamp electrophysiology studies were conducted to identify Kv7.2. Kv7.2/3, Kv7.4, and Kv7.5 selectivity. Our results validate the SyncroPatch platform and establish new structure activity relationships (SAR). Specifically, in addition to selective Kv7.2, Kv7.2/3, Kv7.4. and Kv7.5 agonists, we identified a novel chemotype, ZK-21, a 4-aminotetrahydroquinoline that is distinct from any of the previously described Kv7 channel modifiers. Using flexible receptor docking, ZK-21 was predicted to be stabilized by W236 and bind perpendicular to retigabine, burying the benzyl carbamate group into a tunnel reaching the core of the pore domain.
Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose–response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC50 for hERG inhibition is estimated from diverse historical proprietary data. The IC50 derived from a two-point proprietary screening data set demonstrated high correlation (R = 0.98, MAE = 0.08) with IC50s derived from six-point dose–response curves. Similar IC50 estimation accuracy was obtained on a public thallium flux assay data set (R = 0.90, MAE = 0.2). The IC50 data were used to develop a robust quantitative model. The model’s MAE (0.47) and R2 (0.46) were on par with literature statistics and approached assay reproducibility. Using a continuous model has high value for pharmaceutical projects, as it enables rank ordering of compounds and evaluation of compounds against project-specific inhibition thresholds. This data inference approach can be widely applicable to assays with quantitative readouts and has the potential to impact experimental design and improve model performance, interpretation, and acceptance across many standard safety endpoints.
Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose–response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC50 for hERG inhibition is estimated from diverse historical proprietary data.
In our continuing efforts to discover novel triazoles with improved antifungal activity in vitro and in vivo, a series of 41 novel compounds containing 1,2,3-triazole side chains were designed and synthesized via a click reaction based on our previous work. Most of the compounds showed moderate to excellent broad-spectrum antifungal activity in vitro. Among them, the most promising compound 9A16 displayed excellent antifungal and anti-drug-resistant fungal ability (MIC80 = 0.0156–8 μg/mL). In addition, compound 9A16 showed powerful in vivo efficacy on mice systematically infected with Candida albicans SC5314, Cryptococcus neoformans H99, fluconazole-resistant C. albicans 100, and Aspergillus fumigatus 7544. Moreover, compared to fluconazole, compound 9A16 showed better in vitro anti-biofilm activity and was more difficult to induce drug resistance in a 1 month induction of resistance assay in C. albicans. With favorable pharmacokinetics, an acceptable safety profile, and high potency in vitro and in vivo, compound 9A16 is currently under preclinical investigation.
4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.
Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with GΩ seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1,920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPRTM. Additionally, we tested the effect of 36 arthropod venoms on NaV1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through FACS prior to APC experiments. GΩ seal data quality makes the 384- format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, this highlights the broad applicability and versatility of APC platforms and also outlines some limitations of the approach.
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.
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.
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.
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.
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 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’ 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.
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.
Transient Receptor Potential Canonical (TRPC) channels are a subfamily of TRP channels. The TRPC family contains at least 7 subunits and are predominantly expressed in neuronal cells where they may play an important role in Ca2+ flux. TRPC5 channels are non-selective cation channels expressed in many areas of the brain particularly the hippocampus, amygdala and cerebellum, amongst others. Although the physiological and pathophysiological role of TRPC5 is not fully known, it does appear to be important in neuronal function, in particular during development where it is involved in hippocampal neurite outgrowth and growth cone morphology. Knockout mouse studies have also revealed that TRPC5 plays an essential role in innate fear. TRPC5 is expressed in some regions outside of the CNS including the heart where it contributes to cardiac hypertrophy in heart failure. TRPC5 is activated by intracellular calcium. Using the SyncroPatch 384PE, TRPC5 expressed in HEK cells could be activated by perfusion of the intracellular solution to contain free-Ca2+. This current was potentiated by the lanthanide, gadolinium (Gd3+), as expected and blocked by 2-APB with an IC50 consistent with that reported in the literature.
Transient receptor potential (TRP) channels have become important potential targets in drug discovery for the treatment of, for example, pain, respiratory diseases such as asthma, cancer and immune disorders, multiple kidney diseases and skeletal disorders1 . The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP family of cation channels, plays a predominant role in the sensation of noxious cold2 and inflammatory pain3 . The channel is activated by a range of environmental irritants causing pain, pungent compounds found in foods such as garlic, mustard and cinnamon, as well as metabolites produced during oxidative stress4 . Consistent with its proposed function in nociception, TRPA1 has been shown to be expressed in sensory neurons of the dorsal root ganglion (DRG) and trigeminal ganglion, both of which transmit painful responses2. Thus, within drug development, much attention is paid to the TRPA1 channel. Preclinical data and data from a recent human genetic study5 highlight TRPA1 antagonists as a promising new approach for the treatment of acute and chronic pain. Indeed, a TRPA1 antagonist has shown positive results in a proof-ofconcept study for diabetic neuropathic pain6 . The most challenging aspects involved in the screening of the TRPA1 channel are the channel’s mechanosensitivity, fast desensitization and activity dependence on intracellular calcium. Here, we present high quality data with reliable pharmacology on hTRPA1 expressing HEK cells collected on the SyncroPatch 384PE. Data is presented showing activation of the TRPA1 channel by SCMA and inhibition by A-967079.
The TWIK-related K+ channel (TREK-1) is a two pore domain (K2P) K+ channel encoded by the KCNK2 gene. The protein is a homodimer, each subunit comprised of transmembrane (TM) helical regions (M1-M4), two pore domains (P1 & P2), an extracellular region with 2 helices, and intracellular N and C termini. The pore helices and pore loops form the K+ selectivity filter. First discovered in 1996, it plays an important physiological role in background K+ conductance and thus plays a major role in regulating resting membrane potential. TREK-1 widely is expressed throughout the CNS and spinal cord, particularly in the cortex, hippocampus, thalamus, hypothalamus, cerebellum and basal ganglia. Additionally, TREK-1 is expressed in high levels in small and medium sized dorsal root ganglion (DRG) neurons. It is also expressed in other regions such as lung, heart, kidney, skeletal muscle and human myometrium where it is up-regulated during pregnancy5 and may play a role in maintaining a negative membrane potential prior to labor. TREK-1 is modulated by a variety of different physical and chemical stimuli including mechanical (stretch), temperature, intracellular acidosis, poly-unsaturated fatty acids and phospoholipids. It has also been shown to be opened by volatile anaesthetics and is likely to be an important target for these agents. TREK-1 has been proposed to play a pivotal role in cerebral ischemia, epilepsy, depression, pain perception and temperature sensing and is an interesting therapeutic target. Human TREK-1 expressed in HEK cells were recorded on the SyncroPatch 384PE with good success rates. hTREK-1 was activated by BL-1249 and blocked by THA.
TMEM16A/Anoctamin1 is a Ca2+-activated Cl− channel (CACC) which has a broad functional spectrum in processes including trans-epithelial ion transport, olfaction, photo-transduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. TMEM16A has been implicated to play a role in a number of health disorders and may be an important therapeutic target in cystic fibrosis, asthma, pain and some human cancers. TMEM16A is activated by elevated cytosolic calcium concentrations. In conventional patch clamp experiments, exchange of the intracellular solution to include calcium in order to initiate channel activity is challenging and typically performed using inside-out patches or by comparing the effect of internal calcium between different cells in the whole cell configuration. Furthermore, current run-down or desensitization are common problems associated with recording this ion channel. Here, we present data from HEK293 cells expressing hTMEM16A in whole cell and perforated patch mode using fluoride-free internal solution on the SyncroPatch 384PE. The data show that intracellular solution can be exchanged in a very robust manner to investigate calcium sensitivity, voltage dependence and pharmacology of the channel.
The resting membrane potential of excitable cells is determined by leak conductances predominantly mediated by KCNK and two-pore-domain potassium channels (K2P). K2P channels are characterized by the presence of two pore forming regions and four trans-membrane spanning (4TMS) regions in each channel subunit and form functional dimers. These channels are essential for the production of background leak type potassium currents that act to regulate resting membrane potential and levels of cellular excitability. The TWIKrelated acid-sensitive K+ channel 1 (K2P3.1 or TASK-1) is a member of the K2P channel family and is encoded by the KCNK3 gene. TASK-1 is ubiquitously expressed throughout the CNS but also in other tissues such as in the heart, adrenal gland, lung, pancreas,kidney, intestine and prostate. TASK-1 has been implicated in atrial fibrillation (AF) pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. TASK-1 is activated by extracellular acidosis and inhibited by anandamide and by local anesthetics including bupivicaine. Volatile general anestethics such as halothan and xenon stimulate TASK-1.
Recombinant cell lines that functionally express human cardiac ion channels are a valuable tool for testing new drugs for potential side effects that induce proarrhythmia. It can be difficult to maintain a constant quality of these cell lines in a continually passaged culture making this process incompatible with routine screening in high-throughput mode. Here we demonstrate the preparation of Patch Ready Cells prepared from five cell lines expressing recombinant ion chan-nels (B’SYS, Switzerland) which are recommended by the CiPA initiative for drug safety testing. The Patch Ready Cells have been tested by automated patch-clamp on a SyncroPatch 384PE (Nanion, Germany) to demonstrate their applicability in high-throughput cardiotoxicity testing.
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. Additionally, 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 SyncroPatch 384PE showing activation and inhibition of P2X3 currents expressed in CHO cells with rapid and brief application of ligand (‘Ligand Puff’). ATP or αβ-methylene ATP (αβ-MeATP) activated P2X3 receptors with an EC50 value similar to values found in the literature. P2X3 receptors could be repetitively activated by ATP and blocked by A-317491 with an IC50 value in good agreement with the literature.
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 the SyncroPatch 384PE showing activation and inhibition of P2X2/3 currents expressed in CHO cells with rapid and brief application of ligand (‘Ligand Puff’). 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. P2X2/3 receptors could be repetitively activated by ATPand blocked by suramin with an IC50 in good agreement with the literature.
N-Methyl-D-aspartate (NMDA) receptors are a member of the ionotropic glutamate receptor family, ligandgated ion channels that mediate the majority of excitatory neurotransmission in the mammalian CNS. They are expressed primarily in the CNS but also in peripheral locations such as pancreatic islet cells, sensory nerve terminals in skin and cardiac ganglia. Seven subunits of the NMDA receptor have been identified, NR1, NR2A-D and NR3A-B2 , they assemble as a tetramer consisting of two NR1 subunits and either two NR2 subunits or a combination of NR2 and NR3 subunits. Activation of NMDA receptors requires the simultaneous binding of glutamate and glycine. Calcium entry through NMDA receptors plays an important role in development and synaptic plasticity and is proposed to underlie higher processes such as learning and memory. It is also proposed to play a role in a number of neurological diseases such as epilepsy and Alzheimer’s. Indeed, memantine is an NMDA antagonist which has been approved for the treatment of moderate to severe Alzheimer’s. NMDA antagonists may also be targets for the treatment of neuropathic pain, major depression and Parkinson’s disease. Here we present high quality data at a high throughput collected on the SyncroPatch 384PE showing activation and inhibition of NMDA NR1/ NR2B expressed in HEK cells. Stable recordings of NMDA receptor were achieved and modulation of the response by spermine and ketamine is shown.
Human neurons derived from induced pluripotent stem cells (hiPSCs) are becoming increasingly important for studying basic neuronal physiology and can provide good models for studying neurological disorders. hiPSC derived neurons provide a viable alternative to primary cells and animal models in the drug discovery industry for finding novel therapeutics to treat seizure-related and neurodegenerative disorders. iCell® GlutaNeurons are glutamatergic-enriched cortical neurons derived from hiPSCs. Single cell gene transcription analysis has shown the presence of glutamate receptors: AMPA, kainate and NMDA, as well as glutamate and GABA transporters. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the mammalian CNS and removal of glutamate from the synaptic cleft by reuptake via glutamate transporters plays a role in regulating neuronal excitability. GABA is the major inhibitory neurotransmitter in the brain and is important in controlling excitability. After release, GABA is removed from the extracellular space by GABA transporters(GATs), thus terminating inhibitory synaptic transmission. Both GABA and glutamate transporters may provide novel therapeutic targets for, e.g. Parkinson’s disease, Alzheimer’s disease, and epilepsy.We recorded ligand-gated ion channel currents mediated by GABAA and AMPA receptors from iCell® GlutaNeurons on the Patchliner and SyncroPatch 384PE. Furthermore, we could measure GABA and glutamate transporters in these neurons using the SURFE2R N1 device.
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. In contrast to the fast and rapidly inactivating TTX-sensitive channels, NaV1.8 is TTX resistant and exhibits slower kinetics with a depolarized voltage-dependence of activation and inactivation. hNaV1.8 is an interesting drug target for inflammatory and neuropathic pain because modulation of this ion channel by inflammatory mediators appears to be a key mechanism of DRG nociceptor sensitization and activation. Interestingly, the development of potent and selective NaV1.8 inhibitors has shown promising results in reducing neuropathic pain in animal models and this has fueled interest in the search for selective NaV1.8 inhibitors. The bottleneck for drug discovery involving ion channels is often the electrophysiological assays. Nanion’s SyncroPatch 384PE offers a high throughput gigaseal platform which records up to 384/768 experiments simultaneously which helps to address this problem. It enables the recording of high quality data with reliable pharmacology, and biophysical characterizations of the protein. Our results show current-voltage relationships consistent with published results and very stable recordings using multi-hole chips. Furthermore, we show activation of hNaV1.8 from different states results in altered compound affinity. We demonstrate the suitability of Nanion’s SyncroPatch 384PE for high throughput screening of hNaV1.8.
Automated patch clamp (APC) instruments are used for a wide variety of applications ranging from basic research into channelopathies and biophysical characteristics of ion channels, through to routine cardiac safety testing. Their use 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. It is well known that fluoride is often used in the internal solution in APC experiments to improve the seal resistance. The presence of external calcium (or other divalent cation) further improves the seal by a mechanism thought to be due to the formation of CaF2 crystals at the interface between the pipette or micro-pore and the cell as described in a recent patent application.Even in manual patch clamp experiments, fluoride has been used to record voltage gated Na+ channels for over 20 years, despite known effects on voltage dependence of conductance, and steady-state fast inactivation and its inhibition of protein phosphatase. Fluoride is used because it improves the seal and allows stable measurements to be performed over long periods of time. However, because there are some experiments where it is advantageous to use physiological, fluoride-free internal solutions and external solution that does not use divalent ‘seal enhancer’ solutions, we have developed a method that allows fluoride-free, physiological solutions to be used with good success rates. We demonstrate this using the cardiac ion channels hERG expressed in HEK293 cells (SB Drug Discovery) and NaV1.5 expressed in CHO cells (Charles River).
The NaV1.7 gene (SCN9A) encodes a voltage-gated sodium (NaV) channel, primarily expressed in the peripheral nervous system. It 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 part 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 exhibit both state- and use-dependence. For this reason, it is important to be able to reliably measure the effects of compounds using different voltage protocols to investigate state and use-dependency. In this Application Note we present data using the SyncroPatch 384PE characterizing CHO cells stably expressing hNaV1.7. The current-voltage relationship and the state- and use-dependence effects of the sodium channel blocker, tetracaine, are shown.
High throughput screening (HTS) is used in the pharmaceutical industry to aid drug discovery. Large numbers of chemical compounds can be tested for biological activity using a range of techniques. The patch clamp technique remains the gold standard to test activity of compounds on ion channels and automated patch clamp (APC) is increasingly adopted in HTS labs as an alternative to conventional patch clamp given its increased ease-of-use and higher throughput. APC is employed in all aspects of drug discovery from hit finding and lead optimization through to target validation and safety testing. This is only possible due to the increase in throughput toward HTS capabilities, the compatibility with HTS workflows, and a lower cost per data point which can compete with other techniques such as fluorescence imaging (using, for example, the FLIPRTM instrument) and calcium imaging with the added benefit of real-time kinetics of drug effects. Indeed, all the major contract research organizations worldwide use APC for ion channel screening and cardiac safety testing. Increased automation, including unattended operation, is also an important factor for increasing throughput, and instruments can reliably work beyond an 8-h day provided they are serviced with enough cells, solutions, and compounds. For this to work effectively, data must be reliable with high success rates, low false positive and negative rates along with reproducible IC50 values.
Nicotinic Acetylcholine Receptors (nAChR) are cationpermeable ion channels, which mediate fast synaptic transmission when activated by the endogenous neurotransmitter acetylcholine (ACh) and the exogenous natural alkaloid, nicotine. Neuronal nAChR form pentameric channels which are composed of two α (α2 to α10) and three β subunits (β2 to β4). Mutations of nAChR are associated with some forms of epilepsy and many other neurological disorders such as Alzheimer’s Disease, Parkinson’s, Tourette’s Syndrome, Schizophrenia and depression. The most abundantly expressed nAChR in the mammalian brain are the α7 homomeric and α4β2 heteromeric receptors. In contrast to the α7, a4β2 nAChR has a high affinity for nicotine. This property, the up-regulation during chronic exposure to nicotine, and the receptor expression location in addiction sensitive regions of the brain like the ventral tegmental area, strongly indicate that the a4β2 nAChR is a potential target for addiction to nicotine. Here we present data collected on the SyncroPatch 384PE showing activation and block of α4β2 nAChR currents expressed in HEK cells with rapid application of ligand (‘Ligand Puff’). ACh activates α4β2 nAChR in a concentration dependent manner with an EC50 value similar to those reported in the literature. Reproducible currents were achieved when cells were preincubated with acetylcholinesterase (AChE). Finally, α4β2 nAChR were blocked by dihydro-b-erythroidine hydrobromide (DHßE), a well known competitive antagonist of the α4 subunit3 with an IC50 in good agreement with the literature.
The transient receptor potential ankyrin 1 (TRPA1) is a calcium permeable non-selective cation channel that belongs to the transient receptor potential (TRP) superfamily. The TRPA1 channel is expressed in the sensory neurons of the nodose ganglia, dorsal root ganglia, and trigeminal ganglia, and also non-neuronal cells such as cardiomyocytes, lung fibroblasts and pancreatic β cells. TRPA1 is activated by a range of natural pungent compounds including allyl isothiocyanate (AITC),cinnamaldehyde, and allicin. TRPA1 can also be activated by cold temperature and has been proposed to act as a mechanosensor. Not only has TRPA1 been proposed to play a role in nociception and certain pain conditions, but has also in cardiovascular conditions such as atherosclerosis, heart failure, arrhythmia, vasodilation, and hypertension. Thus, within drug development, much attention is paid to the TRPA1 channel. For example, TRPA1 has been identified as a potential target for persistent chronic painful states including inflammation, neuropathic pain, diabetes, fibromyalgia, bronchitis, and emphysema. Indeed, the TRPA1 antagonist GR 17536 from Glenmark showed efficacy in a Phase IIa proof-of-concept clinical trial for peripheral diabetic neuropathy.
The SCN11A gene encodes the voltage-gated sodium channel NaV1.9 which is predominantly expressed in small-diameter sensory neurons of dorsal root ganglia (DRG) and trigeminal ganglia. NaV1.9 is characterized by slow activation with little depolarization near the resting membrane potential generating a persistent, tetrodotoxin (TTX)-insensitive current which inactivates only slowly. These properties suggest that the conductance mediated by NaV1.9 mainly contributes to amplification of depolarizing responses to subthreshold stimuli leading to lower action potential (AP) firing thresholds and increase in AP firing frequency. The role of hNaV1.9 has yet to be fully elucidated but is proposed to be involved in nociception of inflammatory and neuropathic pain. Several gain-of function mutations in SCN11A have been identified which result in either painful neuropathy or an insensitivity to pain. Given its proposed role in pain perception, NaV1.9 has gained some attention as a potential target for the development of novel pain therapeutics. Here we present high quality data with reliable pharmacology on hNaV1.9 expressing HEK293 cells at a high throughput collected on the SyncroPatch 384. Biophysical properties of NaV1.9 expressed in HEK cells (cells kindly provided by Icagen, Inc., USA) and concentration response curves for three NaV channel blockers are shown, including use-dependence of tetracaine.
Neuronal and cardiac rhythmicity is predominantly controlled by hyperpolarization activated cyclic nucleotide gated (HCN) channels. The HCN family comprises four members (HCN1-4) which are ubiquitously expressed in the central and peripheral nervous system. Activated by hyperpolarization, HCN channels open slowly with no inactivation. Cyclic AMP (and other second messenger proteins) affects the activation properties independent of phosphorylation, modulating the voltage dependence of current activation and accelerating the kinetics of channel opening. HCN mediates a Na+/K+ conductance (Ih) which contributes to the establishment of the resting membrane potential. It is therefore not surprising that HCN channels play an important role in the regulation of neuronal firing and excitability as well as pacemaking. Disruption of HCN function slows down the heart rate and provides a potential target for the treatment of neuronal disorders such as epilepsy and neuropathic pain.Here we present data collected on the SyncroPatch 384PE showing pharmacology and modulation of hHCN2 expressed in HEK cells. In addition, these cells heterologously express a light-sensitive bacterial phospho-adelynate cyclase (bPAC). We demonstrate two ways of triggering the cAMP pathway in order to modulate the HCN2 channel opening kinetics. First, we used the internal perfusion system of the SyncroPatch 384PE for direct application of cAMP to the intracellular environment. Second, we triggered the cAMP pathway by optical stimulation of bPAC. Further, we showed voltage dependent block of Ih with Cs+ and ZD7288. Ivabradine, a drug used for symptomatic management of stable heart related chest pain and heart failure blocked the channel with an IC50 of 0.1 mM in good agreement the literature.
The hERG gene encodes a potassium channel responsible for the repolarization of the IKr current in cardiac cells. 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 antiarrhythmias and anti-psychotics, it has become an important target in compound safety screening. A large range of therapeutic agents with diverse chemical structures have been reported to induce long QT syndrome by inhibiting the hERG channel. These include antihistamines (e.g. Terfenadine), gastrointestinal prokinetic agents (e.g. Cisapride), amongst others. Therefore, it is important to test new therapeutics for actions on the hERG channel early on in the drug discovery process. Here we present high quality data with reliable pharmacology on hERG expressing CHO cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots, and concentration response curves for the compounds pimozide, astemizole, cisapride and terfenadine are shown. The IC50 values for these compounds are within the expected range and success rates of 80% for completed experiments were recorded.
The voltage-gated chloride channel gene (ClC) family is highly conserved and their members are present in both pro- and eukaryotes. In mammals, nine different ClC subtypes have been identified, which differ according to tissue distribution and subcellular location. ClC-1 is exclusively expressed in skeletal muscles. ClCs function as homodimers, allowing chloride and other anions to be conducted through each single protopore. Gating of the single monomer is fast while the common gate to open and close the pores simultaneously is slow. The channel gating can be modulated by intracellular and extracellular chloride as well as pH. ClC proteins mediate chloride flux across cellular membranes in most cell types and participate in maintenance of resting membrane potential. Plasma membrane chloride channels play an important role in reducing muscle excitability. ClC-1 contributes to membrane repolarization and stabilizes the membrane voltage in skeletal muscle. Experimental block of the chloride conductance mediated by ClC-1 facilitated muscle hyperexcitability, manifested as myotonia. Here we present data conducted on the SyncroPatch 384 showing characteristic biophysical properties and pharmacology of hClC-1 expressed in CHO cells. We applied voltage protocols including various test potentials to study the voltage dependence of compounds. In order to investigate compound binding properties we used the internal perfusion system of the SyncroPatch 384 for direct application of anthracene-9-carboxylic acid (9-AC) to the intracellular environment. Moreover, we also investigated the effect of 9-AC and niflumic acid (NFA) when they are applied from the extracellular side.
Acid-sensing ion channels (ASICs) are protongated ion channels which are highly sensitive to extracellular acidosis and are permeable to cations, predominantly Na+. They are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG)family.
Cystic Fibrosis (CF) is an autosomal recessive genetic disorder which affects a number of organs, in particular the lungs, pancreas and sweat glands. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR). CFTR is a regulated epithelial chloride channel and mutations cause a reduction in activity of the channel via a variety of mechanisms. This results in defective electrolyte transport in airway epithelia and thereby, chronic lung infection and premature mortality. Therefore, compounds which increase activity of CFTR have therapeutic potential for treating CF. The CFTR protein is composed of 5 domains: there are 2 transmembrane (TM) domains, 2 nucleotidebinding domains (NBDs) and 1 regulatory domain (R). The TM domains form the pore of the channel, channel activity is determined by phosphorylation of the R domain and gating is controlled by hydrolysis of ATP at the NBD. CFTR is activated via a number of reagents including internal fluoride, cAMP and external forskolin. Here we show activation of CFTR expressed in CHO cells on the SyncroPatch 384PE by internal perfusion of F- or external application of forskolin. The current was blocked with the specific blocker, inh-172 with an IC50 in good agreement with the literature. In addition, CFTR activated by internal cAMP was potentiated by VX-770. Using F- -free internal solution and activation by submaximal cAMP or forskolin, potentiators of CFTR can be investigated as potential therapeutics to treat CF.
The CaV3.2 channel is one of the three low voltage activated (LVA) T-type calcium channels. The LVA currents differ from the high voltage activated (HVA) calcium currents in their activation and inactivation kinetics. LVA currents are activated at lower voltages (typically activating above -50 mV and peaking at around -20 mV), they display faster inactivation, slower deactivation and a smaller conductance of Ba2+ ions as compared with the HVA currents. The CaV3.2 channel contains the α1H subunit, encoded by the CACNA1H gene on the human chromosome 16p13.3. T-type channels are expressed in a wide variety of organs throughout the human body, including nervous tissue, heart, kidney, smooth muscle, and many endocrine organs. They have been implicated in a variety of physiological processes including neuronal firing, smooth muscle contraction and hormone secretion. More recently, CaV3.2 has been shown to play a role in nociception and pain. Here we present high quality data with reliable pharmacology on CaV3.2 expressing HEK cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nitrendipine, nifedipine, mibefradil and amiloride are shown. The IC50 values for these compounds are within the expected range and success rates of up to 79% for completed experiments were recorded.
The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p133. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, in the light of the CiPA initiative, the L-type channel is likely to become an important target for cardiac safety testing. Here we present high quality data with reliable pharmacology on CaV1.2 expressing CHO cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >70% for completed experiments were recorded. Importantly, CaV1.2 recorded on the Syncro- Patch 384PE exhibited stable peak amplitudes during the course of the experiment and displayed little or no rundown.
The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and neuroendocrine regulation. VGCC are composed of 4 subunits, which include an alpha 1 subunit which forms the Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p13. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, the L-type Ca2+ channel is an important target for cardiac safety testing, especially in the light of the CiPA initiative. Here we present high quality data with reliable pharmacology on Assay Ready CaV1.2 Cells at a high throughput collected on the SyncroPatch 384. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >75% for completed experiments were recorded. Importantly, CaV1.2 recorded on the Syncro- Patch 384 exhibited stable peak amplitude during the course of the experiment and displayed low rundown.
The CaV1.2 channel is a voltage-gated calcium channel (VGCC) expressed in a variety of mammalian tissues and is essential for multiple processes including CNS function, cardiac and smooth muscle contraction and neuroendocrine regulation. VGCC are composed of 4 subunits, which include an alpha 1 subunit which forms the Ca2+-selective pore, contains the voltage sensor and many of the binding sites for regulatory modulators and drugs and accessory subunits α2δ, β and γ which are involved in anchorage, trafficking and regulatory functions. The CaV1.2 channel contains the alpha-1C subunit, encoded by the CACNA1C gene on the human chromosome 12p13. Mutations in the L-type Ca2+ channels have been associated with inherited arrhythmic disorders such as Timothy, Brugada and early repolarization syndromes. In addition, in the light of the CiPA initiative, the L-type channel is likely to become an important target for cardiac safety testing. Here we present high quality data with reliable pharmacology on CaV1.2 expressing HEK cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for the compounds nifedipine and verapamil are shown. The IC50 values for these compounds are within the expected range and success rates of >75% for completed experiments were recorded. Importantly, CaV1.2 recorded on the SyncroPatch 384PE exhibited stable peak amplitude during the course of the experiment and displayed little or no rundown.
Looking for new targets in pain and cancer therapy, the transient receptor potential chan-nels (TRP-channels) gained a lot of interest during the last decade. The ligand-gated calcium channels play an important role in the perception of pain and temperature and are often dysregulated in tumor tissues. They have become appealing targets for Drug Discovery. Re-combinant cell lines which stably express different TRP-channels have been successfully used for lead identification and compound profiling.Looking for new targets in pain and cancer therapy, the transient receptor potential chan-nels (TRP-channels) gained a lot of interest during the last decade. The ligand-gated calcium channels play an important role in the perception of pain and temperature and are often dysregulated in tumor tissues. They have become appealing targets for Drug Discovery. Re-combinant cell lines which stably express different TRP-channels have been successfully used for lead identification and compound profiling.Assay ready cryopreserved aliquots prepared from these cell lines can be used instantly after thawing without prior cultivation. Here, we demonstrate that Assay Ready Cells prepared from TRP-channel expressing cell lines resemble the pharmacology of cells from continuous culture in different end-point assays. The cells were successfully qualified for plate-based fluorescent calcium-flux assays and for recording of activated ion channel currents using automated patch clamping.
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 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. Here we present data collected on the SyncroPatch 384PE 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.
Acid-sensing ion channels (ASICs) are ligand-gated ion channels activated by protons. They are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family. ASICs are highly sensitive to extracellular acidosis and are permeable to cations, predominantly Na+. So far, 6 different ASIC subunits have been identified encoded by 4 genes. They are found expressed throughout the CNS and PNS and have a proposed role in nociception and pain, and other neurological diseases such as ischaemia and inflammation. The ASIC3 channel was first identified in the late 1990’s. It was found to be localized to primary afferent nociceptive fibers innervating the skin, muscles, joints and viscera, in agreement with a role in pain perception. Furthermore, ASIC3 is expressed in higher amounts in nociceptive neurons innervating muscle (~ 50%) compared to skin (~ 10%), which indicates that ASIC3 may play an important role in detecting muscle acidosis. Here we present high quality data at a high throughput collected on the SyncroPatch 384PE showing activation and inhibition of ASIC3 expressed in HEK cells. The pH which elicited a halfmaximal response was in good agreement with the literature. The IC50 for block of the ASIC3 current by amiloride, a known blocker of ASIC and ENaC channels, was also in good agreement with the literature. Success rates of over 80% for completed experiments were recorded.
The cardiac late Na current (late INa) generates persistent currents throughout the plateau phase of the cardiac action potential. Several mutations in the SCN5A gene cause a form of hereditary long QT syndrome (LQT3)1-3. The ΔKPQ mutation deletes residues Lys 1505, Pro 1506 and Gln 1507, resulting in a sustained, non-inactivating current during long (over 50 ms) depolarizations1,2. This sustained current causes prolongation of the action potential which can result in fatal ventricular arrhythmias such as Torsade de Pointes (TdP)1.One aim of the Comprehensive In Vitro Pro-arrhythmia Assay (CiPA) initiative is to improve drug safety testing in pre-clinical development by evaluating the pro-arrhythmic risk of a compound4,5. Validation studies confirm that testing the effect of compounds on an increased number of human cardiac ion channel currents including INa (NaV1.5 peak and late current) as well as IKr (hERG) leads to improved prediction of their clinical risk. Late INa can be recorded in WT NaV1.5 channels using the toxin ATX-II or veratridine, or using a cell line with LQT3 mutations in NaV1.5 without the need for pharmacological enhancement. The latter might also reduce the risk of cross-reactions between late-current enhancers and test compounds.Here we present data collected on the Syncro- Patch 384i showing the peak and late INa current re¬corded from WT and NaV1.5-ΔKPQ cell lines. Peak current could be reliably recorded from both cell types. In WT cells, late INa was negligible in the absence of ATX-II, whereas the late INa from NaV1.5-ΔKPQ cells could be reliably recorded. Peak current from WT, and peak and late INa from NaV1.5-ΔKPQ was inhibited by ranolazine and mexiletine and IC50 values agreed well with the literature6.
Nicotinic acetylcholine receptors (nAChR) are acetylcholine- (ACh) and nicotine-gated cation permeable ion channels, which mediate fast synaptic transmission at central synapses and neuromuscular junctions. Neuromuscular nAChR form heteromeric proteins composed of four subunits: α, β, γ (or ε) and δ. Depending on the developmental stage, the AChR subunit stoichiometry changes from α1β1γδ (embryonic) to α1β1εδ (adult). Several inherited and acquired diseases are associated with nAChR dysfunction, most of which lead to impaired neuromuscular transmission and muscle weakness. The acquired autoimmune disease myasthenia gravis (MG) is caused by autoantibodies targeting muscle nAChRs that disrupts nerve-muscle communication resulting in muscle weakness and fatigue. Inherited diseases called congenital myasthenic syndromes (CMS) are associated with several abnormalities affecting ACh-release, acetylcholinesterase activity, nAChR function and/or nAChR number. Treatment has been limited to nonselective, chronic immunosuppressive therapies which have longterm toxicities. More selective and targeted therapies are now under development. Here we present data collected on the SyncroPatch 384 showing activation and block of nAChRα1β1γδ expressed in human TE671 cells with rapid application of ligand or co-application with blockers. We found that ACh activates nAChRα1β1γδ receptors with an EC50 value similar to those reported in the literature. We recorded highly reproducible currents in response to ACh and obtained IC50 values for mecamylamine and α-conotoxin GI that were in good agreement with the literature.
The cardiac action potential is defined by multiple voltage-dependent ion channels. A drug candidate’s capacity to interact with the ion channels involved in the depolarization or repolarization phases of the cardiac action potential is important for drug safety assessment. Until now, 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. A new paradigm, the Comprehensive In-vitro Proarrhythmia Assay (CiPA), was introduced in 2013 to provide a more complete assessment of proarrythmic risk. An assessment of a multitude of cardiac ion channels, in addition to hERG, should provide a more accurate prediction of the proarrythmic risk of a compound compared with testing on hERG alone. Here we show recordings from HEK or CHO cells expressing the CiPA stipulated ion channels; NaV1.5, CaV1.2, hERG, KV7.1, Kir2.1 or KV4.3, activated within one single experiment on the SyncroPatch 384PE.
The NaV1.5 channel, encoded by the SCN5A gene, is a voltage-gated sodium (NaV) channel found in skeletal muscle and heart. It is TTX insensitive with an IC50 in the micromolar range. NaV1.5 is responsible for the upstroke of the cardiac action potential in both ventricular and atrial myocytes and is therefore critical for generation and propagation of the cardiac action potential in human heart. Block of this channel can lead to prolongation of the QRS interval of the electrocardiogram (ECG) and can have profound effects on the rate of cardiac deploarization and conduction velocity, thus causing potentially dangerous cardiac arrythmias. Furthermore, effects of NaV1.5 inactivation can modify cardiac repolarization. Given the importance of this channel in maintaining cardiac function, it has become an important target in compound safety screening. Local anaesthetics, such as lidocaine, have been shown to exhibit state- and use-dependence when acting on the cardiac sodium channel. The IC50 was shown to be approximately 30 times lower at depolarized holding potentials where inactivation was almost complete. For this reason, it is important to test potency of compounds at different holding potentials. Here we present high quality data with reliable pharmacology on hNaV1.5 expressing HEK293 cells at a high throughput collected on the SyncroPatch 384PE. Current-voltage plots and concentration response curves for four NaV channel blockers are shown, including lidocaine at different holding potentials.
Abstract: Mutations in the KCNH2 gene are a well-established cause of sudden cardiac death, resulting from disturbed electrical signalling, in otherwise healthy young people. Yet, the majority of missense variants identified in KCNH2 are likely to be benign. To differentiate between benign and pathogenic variants in KCNH2 we have developed a high throughput functional assay using the syncropatch 384PE automated patch clamp system. The Victor Chang Cardiac Research Institute is an independent, not-for-profit, medical research facility that is dedicated to finding cures for cardiovascular disease. Renowned for the quality of its breakthroughs, the Victor Chang Cardiac Research Institute is one of the most respected heart research facilities in the world.
This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.
Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.
SMN protein deficiency causes motoneuron disease spinal muscular atrophy (SMA). SMN-based therapies improve patient motor symptoms to variable degrees. An early hallmark of SMA is the perturbation of the neuromuscular junction (NMJ), a synapse between a motoneuron and muscle cell. NMJ formation depends on acetylcholine receptor (AChR) clustering triggered by agrin and its co-receptors lipoprotein receptor-related protein 4 (LRP4) and transmembrane muscle-specific kinase (MuSK) signalling pathway. We have previously shown that flunarizine improves NMJs in SMA model mice, but the mechanisms remain elusive. We show here that flunarizine promotes AChR clustering in cell-autonomous, dose- and agrin-dependent manners in C2C12 myotubes. This is associated with an increase in protein levels of LRP4, integrin-beta-1 and alpha-dystroglycan, three agrin co-receptors. Furthermore, flunarizine enhances MuSK interaction with integrin-beta-1 and phosphotyrosines. Moreover, the drug acts on the expression and splicing of Agrn and Cacna1h genes in a muscle-specific manner. We reveal that the Cacna1h encoded protein CaV3.2 closely associates in vitro with the agrin co-receptor LRP4. In vivo, it is enriched nearby NMJs during neonatal development and the drug increases this immunolabelling in SMA muscles. Thus, flunarizine modulates key players of the NMJ and identifies CaV3.2 as a new protein involved in the NMJ biology.
Gamma-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Here, we identify a so far undescribed function of β-subunit homomers as proton-gated anion channels. Mutation of a single H267A in β3 subunits completely abolishes channel activation by protons. In molecular dynamic simulations of the β3 crystal structure protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit leading to a pore stabilising ring formation and accumulation of Cl- within the transmembrane pore. Conversion of these residues in proton insensitive ρ1 subunits transfers proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH changes (pH50 is in the range 6- 6.3) and kinetic studies suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus as potential novel drug target.
The cardiac sodium ion channel (NaV1.5) is a protein with four domains (DI-DIV), each with six transmembrane segments. Its opening and subsequent inactivation results in the brief rapid influx of Na+ ions resulting in the depolarization of cardiomyocytes. The neurotoxin veratridine (VTD) inhibits NaV1.5 inactivation resulting in longer channel opening times, and potentially fatal action potential prolongation. VTD is predicted to bind at the channel pore, but alternative binding sites have not been ruled out. To determine the binding site of VTD on NaV1.5, we performed docking calculations and high-throughput electrophysiology experiments. The docking calculations identified two distinct binding regions. The first site was in the pore, close to the binding site of NaV1.4 and NaV1.5 blocking drugs in experimental structures. The second site was at the “mouth” of the pore at the cytosolic side, partly solvent-exposed. Mutations at this site (L409, E417, and I1466) had large effects on VTD binding, while residues deeper in the pore had no effect, consistent with VTD binding at the mouth site. Overall, our results suggest a VTD binding site close to the cytoplasmic mouth of the channel pore. Binding at this alternative site might indicate an allosteric inactivation mechanism for VTD at NaV1.5.
The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. To enable the application of this approach at scale this requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including automated quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8%. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With appropriate consideration of protocol design, including sweep length, number of repetitions, and leak correction, this protocol can be applied on APC to acquire data comparable to manual patch clamp.
Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.
The presence of the Transient Receptor Potential channel Vanilloid type 2 (TRPV2) in red blood cells (RBCs) was recently discovered. TRPV2 is a non-selective cation channel that is reported to be mechanosensitive having numerous properties in common with Piezo1. TRPV2 channels can be activated by Δ9-tetrahydrocannabinol (Δ9-THC) and the changes induced in RBC by application of Δ9-THC can be attributed to TRPV2 channel activity. A previous study shown that addition of 30 µM Δ9-THC led to a large fraction of super hydrated RBCs in a healthy donor consuming marijuana in contrast to significantly milder response from no smoker healthy donors. For further investigations three marijuana consumers with very similar smoking habits were scouted, and blood was collected. Whether this heightened sensitivity of the smokers (MS) vs. no-smokers (NS) RBCs is caused by hypersensitizing of TRPV2 was tackled by comparing MS and NS RBCs in functional patch-clamp measurements using the high - throughput automated patch clamp platform SyncroPatch 384. Whole-cell currents elicited by THC application did not show a significant different thus indicates a similar cellular conductance for MS and NS RBCs and therefore rather a lack of difference in TRPV2 expression level. We demonstrate that APC technology is a suitable tool permits for studying TRPV2 channels in RBCs.
Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.
Slack channels are sodium-activated potassium channels that are encoded by the KCNT1 gene. Several KCNT1 gain of function mutations have been linked to malignant migrating partial seizures of infancy. Quinidine is an anti-arrhythmic drug that functions as a moderately potent inhibitor of Slack channels; however, quinidine use is limited by its poor selectivity, safety and pharmacokinetic profile. Slack channels represent an interesting target for developing novel therapeutics for the treatment of malignant migrating partial seizures of infancy and other childhood epilepsies; thus, ongoing efforts are directed toward the discovery of small-molecules that inhibit Slack currents. This review summarizes patent applications published in 2020–2021 that describe the discovery of novel small-molecule Slack inhibitors.
Cyclic α-aryl β-dicarbonyl derivatives are important scaffolds in medicinal chemistry. Palladium-catalyzed coupling reactions of haloarenes were conducted with diverse five- to seven-membered cyclic β-dicarbonyl derivatives including barbiturate, pyrazolidine-3,5-dione, and 1,4-diazepane-5,7-dione. The coupling reactions of various para- or meta-substituted aryl halides occurred efficiently when Pd(t-Bu3P)2, Xphos, and Cs2CO3 were used under 1,4-dioxane reflux conditions. Although the couplings of ortho-substituted aryl halides with pyrazolidine-3,5-dione and 1,4-diazepane-5,7-dione were moderate, the coupling with barbiturate was limited. Using the optimized reaction conditions, we synthesized several 5-aryl barbiturates as new scaffolds of CaV1.3 Ca2+ channel inhibitors. Among the synthesized molecules, 14e was the most potent CaV1.3 inhibitor with an IC50 of 1.42 μM.
The DNA-PK complex is activated by double-strand DNA breaks and regulates the non-homologous end-joining repair pathway; thus, targeting DNA-PK by inhibiting the DNA-PK catalytic subunit (DNA-PKcs) is potentially a useful therapeutic approach for oncology. A previously reported series of neutral DNA-PKcs inhibitors were modified to incorporate a basic group, with the rationale that increasing the volume of distribution while maintaining good metabolic stability should increase the half-life. However, adding a basic group introduced hERG activity, and basic compounds with modest hERG activity (IC50 = 10–15 μM) prolonged QTc (time from the start of the Q wave to the end of the T wave, corrected by heart rate) in an anaesthetized guinea pig cardiovascular model. Further optimization was necessary, including modulation of pKa, to identify compound 18, which combines low hERG activity (IC50 = 75 μM) with excellent kinome selectivity and favorable pharmacokinetic properties.
Despite the identification of the high incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other two members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout and expression studies in an erythroblast cell line. We report the molecular bases of five Er blood group antigens: the recognised Era, Erb and Er3 antigens; and two novel high incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn (HDFN). Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at −25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to −70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
Authors David J.A. Wyllie and Derek Bowie give a brief history of ionotropic glutamate receptor research and an overview of 5 papers in the 15th January 2022 issue of the Journal of Physiology. This includes the Techniques in Physiology paper by Obergrussberger et al, 2022 highlighting the advances and applications of high throughput methods for automated electrophysiological studies and specifically patch-clamp recording. Automated patch clamp devices such as the SyncroPatch 384 are increasingly becoming workhorses in the pharmaceutical industry as well as small research labs/centres.
Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs.
Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.
Transient Receptor Potential Melastatin 5 (TRPM5) is an intracellular calcium-activated cation-selective ion channel expressed in a variety of cell types. Dysfunction of this channel has recently been implied in a range of disease states including diabetes, enteric infections, inflammatory responses, parasitic infection and other pathologies. However, to date, agonists and positive modulators of this channel with sufficient selectivity to enable target validation studies have not been described, limiting the evaluation of TRPM5 biology and its potential as a drug target. We developed a high-throughput assay using a fluorescent membrane potential dye and a medium- and high-throughput electrophysiology assay using QPatch HTX and SyncroPatch 384PE. By employing these assays, we conducted a primary screening campaign and identified hit compounds as TRPM5 channel positive modulators. An initial selectivity profile confirmed hit selectivity to TRPM5 and is presented here. These small molecule TRPM5 compounds have a high potential both as early tool compounds to enable pharmacological studies of TRPM5 and as starting points for the development of potent, selective TRPM5 openers or positive modulators as novel drugs targeting several pathological states.
Airway epithelial damage is a common feature in respiratory diseases such as COPD and has been suggested to drive inflammation and progression of disease. These features manifest as remodeling and destruction of lung epithelial characteristics including loss of small airways which contributes to chronic airway inflammation. Histone deacetylase 6 (HDAC6) has been shown to play a role in epithelial function and dysregulation, such as in cilia disassembly, epithelial to mesenchymal transition (EMT) and oxidative stress responses, and has been implicated in several diseases. We thus used ACY-1083, an inhibitor with high selectivity for HDAC6, and characterized its effects on epithelial function including epithelial disruption, cytokine production, remodeling, mucociliary clearance and cell characteristics. Primary lung epithelial air-liquid interface cultures from COPD patients were used and the impacts of TNF, TGF-β, cigarette smoke and bacterial challenges on epithelial function in the presence and absence of ACY-1083 were tested. Each challenge increased the permeability of the epithelial barrier whilst ACY-1083 blocked this effect and even decreased permeability in the absence of challenge. TNF was also shown to increase production of cytokines and mucins, with ACY-1083 reducing the effect. We observed that COPD-relevant stimulations created damage to the epithelium as seen on immunohistochemistry sections and that treatment with ACY-1083 maintained an intact cell layer and preserved mucociliary function. Interestingly, there was no direct effect on ciliary beat frequency or tight junction proteins indicating other mechanisms for the protected epithelium. In summary, ACY-1083 shows protection of the respiratory epithelium during COPD-relevant challenges which indicates a future potential to restore epithelial structure and function to halt disease progression in clinical practice.
Hundreds of KCNQ2 variants have been identified by genetic testing of children with early onset epilepsy and/or developmental disability. Voltage-clamp recording from heterologous cells has proved useful for establishing deleterious functional effects of KCNQ2 variants, but procedures adapting these assays for standardized, higher throughput data collection and reporting are lacking. In this study, we employed automated patch clamp recording to assess in parallel the functional and pharmacological properties of 79 missense and 2 in-frame deletion variants of KCNQ2. Among the variants we studied were a training set of 18 pathogenic variants previously studied by voltage-clamp recording, 24 mostly rare population variants, and 39 disease-associated variants with unclear functional effects. Variant KCNQ2 subunits were transiently expressed in a cell line stably expressing KCNQ3 to reconstitute the physiologically relevant channel complex. Variants with severe loss-of-function were also co-expressed 1:1 with WT KCNQ2 in the KCNQ3 cell line to mimic the heterozygous genotype and assess dominant-negative behavior. In total, we analyzed electrophysiological data recorded from 9,480 cells. The functional properties of WT KCNQ2/KCNQ3 channels and pharmacological responses to known blockers and activators determined by automated patch clamp recording were highly concordant with previous findings. Similarly, functional properties of 18 known pathogenic variants largely matched previously published results and the validated automated patch clamp assay. Many of the 39 previously unstudied disease-associated KCNQ2 variants exhibited prominent loss-of-function and dominant-negative effects, providing strong evidence in support of pathogenicity. All variants, exhibit response to retigabine (10 µM), although there were differences in maximal responses. Variants within the ion selectivity filter exhibited the weakest responses whereas retigabine had the strongest effect on gain-of-function variants in the voltage-sensor domain. Our study established a high throughput method to detect deleterious functional consequences of KCNQ2 variants. We demonstrated that dominant-negative loss-of-function is a common mechanism associated with missense KCNQ2 variants but this does not occur with rare population variation in this gene. Importantly, we observed genotype-dependent differences in the response of KCNQ2 variants to retigabine.
Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings.
The transient receptor potential cation channel 5 (TRPC5) plays an important role in numerous cellular processes. Due to this, it has gained considerable attention over the past few years as a potential therapeutic target. Recently, TRPC5 has been shown to be involved in the regulation of podocyte survival, indicating a potential treatment option for chronic kidney disease. In addition, a recent study has shown TRPC5 to be expressed in human sensory neurons and suggests that TRPC5 inhibition could be an effective treatment for spontaneous and tactile pain. To understand these processes more fully, potent and selective tool compounds are needed. Herein we report further exploration of the 2-aminobenzimidazole scaffold as a potent TRPC5 inhibitor, culminating in the discovery of 16 f as a potent and selective TRPC5 inhibitor.
Impaired cerebellar Purkinje neuron firing resulting from reduced expression of large-conductance calcium-activated potassium (BK) channels is a consistent feature in models of inherited neurodegenerative Spinocerebellar Ataxia (SCA). Restoring BK channel expression improves motor function and delays cerebellar degeneration, indicating that BK channels are an attractive therapeutic target. Current BK channel activators lack specificity and potency and therefore are poor templates for future drug development. We implemented an automated patch-clamp platform for high throughput drug discovery of BK channel activators using the Nanion SyncroPatch 384PE system. We screened over 15,000 compounds for their ability to increase BK channel current amplitude under conditions of lower intracellular calcium that is present in disease. We identified several novel BK channels activators that were then re-tested on the SyncroPatch 384PE to generate concentration-response curves (CRCs). Compounds with favorable CRCs were subsequently tested for their ability to improve irregular cerebellar Purkinje neuron spiking, characteristic of BK channel dysfunction in SCA1 mice. We identified a novel BK channel activator, 4-chloro-N-(5-chloro-2-cyanophenyl)-3-(trifluoromethyl)benzene-1-sulfonamide (herein renamed BK-20), that activated BK currents more potently (pAC50 = 4.64) than NS-1619 (pAC50 = 3.7) at a free internal calcium concentration of 270 nM in a heterologous expression system and improved spiking regularity in SCA1 Purkinje neurons. BK-20 had no activity on SK1-3 channels but interestingly was a potent blocker of CaV3.1 (IC50 = 1.05 mM). Our work describes both a novel compound for further drug development in disorders with irregular Purkinje spiking and a unique platform for drug discovery in degenerative ataxias. Significance Statement Motor impairment associated with altered Purkinje cell spiking due to dysregulation of BK expression and function is a shared feature of disease in many degenerative ataxias. BK channel activators represent an outstanding therapeutic agent for ataxia. We have developed a high-throughput platform to screen for BK channel activators and identified a novel compound that can serve as a template for future drug-development for the treatment of these disabling disorders.
Purpose Up to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein NaV1.5. Recent studies suggested that NaV1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant NaV1.5 dominant negative effects and their clinical severity. Results In heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to 75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019). Conclusion Most SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.
Cryptococcus neoformans and Cryptococcus gattii can cause fatal invasive infections, especially in immunocompromised patients. However, few antifungal drugs are available to help treat cryptococcosis. In this study, by compound library screening, we presented the first report of hit compound P163-0892, which had potent in vitro and in vivo antifungal activity against Cryptococcus spp. In vitro tests showed that P163-0892 was not cytotoxic and had highly selective and strong antifungal activities against Cryptococcus spp. with MIC values less than 1 μg/mL. Synergism of P163-0892 and fluconazole was also observed in vitro. The in vivo antifungal efficacy of P163-0892 was assessed in a wax moth larval fungal infection model, and treatment with 10 mg/kg P163-0892 caused a significant reduction in fungal burden and significant extension of the survival time. Taken together, our data indicate that the hit compound P163-0892 warrants further investigation as a novel anti-Cryptococcus agent.
A major advancement has recently occurred in the ability to predict protein secondary structure from sequence using artificial neural networks. This new accessibility to high-quality predicted structures provides a big opportunity for the protein design community. It is particularly welcome for membrane protein design, where the scarcity of solved structures has been a major limitation of the field for decades. Here, we review the work done to date on the membrane protein design and set out established and emerging tools that can be used to most effectively exploit this new access to structures.
Cholesterol is a major regulator of multiple types of ion channels. While there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues, V265 and H222, and high throughput electrophysiology.
α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.
Sequencing Mendelian arrhythmia genes in individuals without an indication for arrhythmia genetic testing can identify carriers of pathogenic or likely pathogenic (P/LP) variants. However, the extent to which these variants are associated with clinically meaningful phenotypes before or after return of variant results is unclear. In addition, the majority of discovered variants are currently classified as variants of uncertain significance, limiting clinical actionability.
Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations.
Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.
As a branch of quantitative systems toxicology, in silico simulations are of growing attractiveness to guide preclinical cardiosafety risk assessments. Traditionally, a cascade of in vitro/in vivo assays has been applied in pharmaceutical research to screen out molecules at risk for cardiac side effects and prevent subsequent risk for patients. Drug cardiosafety assessments typically employ early mechanistic, hazard-oriented in silico/in vitro assays for compound inhibition of cardiac ion channels, followed by induced pluripotent stem cells (iPSCs) or tissue-based models such as the rabbit Purkinje fiber assay, which includes the major mechanisms contributing to action potential (AP) genesis. Additionally, multiscale simulation techniques based on mathematical models have become available, which are performed in silico ‘at the heart’ of compound triage to substitute Purkinje tests and increase translatability through mechanistic interpretability. To adhere to the 3R principle and reduce animal experiments, we performed a comparative benchmark and investigated a variety of mathematical cardiac AP models, including a newly developed minimalistic model specifically tailored to the AP of rabbit Purkinje cells, for their ability to substitute experiments. The simulated changes in AP duration (dAPD90) at increasing drug concentrations were compared to experimental results from 588 internal Purkinje fiber studies covering 555 different drugs with diverse modes of action. Using our minimalistic model, 80% of the Purkinje experiments could be quantitatively reproduced. This result allows for significant saving of experimental effort in early research and justifies the embedding of electrophysiological simulations into the DMTA (Design, Make, Test, Analyze) cycle in pharmaceutical compound optimization.
Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of We identified 35 LoF variants (10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database. detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.
High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch clamp electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.
Many genes, including KCNH2, contain “hotspot” domains associated with a high density of variants associated with disease. This has led to the suggestion that variant location can be used as evidence supporting classification of clinical variants. However, it is not known what proportion of all potential variants in hotspot domains cause loss of function. Here, we have used a massively parallel trafficking assay to characterize all single-nucleotide variants in exon 2 of KCNH2, a known hotspot for variants that cause long QT syndrome type 2 and an increased risk of sudden cardiac death. Forty-two percent of KCNH2 exon 2 variants caused at least 50% reduction in protein trafficking, and 65% of these trafficking-defective variants exerted a dominant-negative effect when co-expressed with a WT KCNH2 allele as assessed using a calibrated patch-clamp electrophysiology assay. The massively parallel trafficking assay was more accurate (AUC of 0.94) than bioinformatic prediction tools (REVEL and CardioBoost, AUC of 0.81) in discriminating between functionally normal and abnormal variants. Interestingly, over half of variants in exon 2 were found to be functionally normal, suggesting a nuanced interpretation of variants in this “hotspot” domain is necessary. Our massively parallel trafficking assay can provide this information prospectively.
Loss-of-function (LOF) variants in the KV11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl+) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z’) scores comparing vehicle to E-4031, a drug that increases KV11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs+) to the Tl+ flux assay buffer resulted in an outstanding Z’ of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase KV11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS KV11.1 variants. We report two new ways to increase target-specific activity in trafficking assays—genetic modification and channel activation—that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic KV11.1 variants.
Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.
Neuropsychiatric disorders such as schizophrenia are challenging to treat due to the biological complexity of the disease and the lack of knowledge of the underlying pathophysiology. Whole exome and genome sequencing studies have identified disease-linked rare variants in patients with large effect size. Here, we functionally characterize the schizophrenia linked variant V1282F in SCN2A, encoding the sodium channel NaV1.2. This variant was introduced into isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs were then differentiated into cortical neurons to understand how the variant and gene may be contributing to disease. We observed a significant (~25%) decrease in sodium current in the V1282F neurons compared to control neurons, suggesting the mutation is causing a loss-of-channel function. These results were supported by recordings in recombinant cells overexpressing either the mutant or wildtype NaV1.2, with the mutant channel having significantly (~75%) lower current amplitude than wildtype. We hypothesize that this phenotype may contribute to disease either through the direct loss of neuronal activity or through subsequent abnormal neurodevelopment.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid–based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
The insecticide deltamethrin of the pyrethroid class mainly targets voltage-gated sodium channels (NaVs). Deltamethrin prolongs the opening of NaVs by slowing down fast inactivation and deactivation. Pyrethroids are supposedly safe for humans, however, they have also been linked to the gulf-war syndrome, a neuropathic pain condition that can develop following exposure to certain chemicals. Inherited neuropathic pain conditions have been linked to mutations in the NaV subtypes NaV1.7, NaV1.8, and NaV1.9. Here, we examined the effect of deltamethrin on the human isoforms NaV1.7, NaV1.8, and NaV1.9_C4 (chimera containing the C-terminus of rat NaV1.4) heterologously expressed in HEK293T and ND7/23 cells using whole-cell patch-clamp electrophysiology. For all three NaV subtypes, we observed increased persistent and tail currents that are typical for NaV channels modified by deltamethrin. The most surprising finding was an enhanced slow inactivation induced by deltamethrin in all three NaV subtypes. An enhanced slow inactivation is contrary to the prolonged opening caused by pyrethroids and has not been described for deltamethrin or any other pyrethroid before. Furthermore, we found that the fraction of deltamethrin-modified channels increased use-dependently. However, for NaV1.8, the use-dependent potentiation occurred only when the holding potential was increased to −90 mV, a potential at which the tail currents decay more slowly. This indicates that use-dependent modification is due to an accumulation of tail currents. In summary, our findings support a novel mechanism whereby deltamethrin enhances slow inactivation of voltage-gated sodium channels, which may, depending on the cellular resting membrane potential, reduce neuronal excitability and counteract the well-described pyrethroid effects on channel activation.
Neurogranin (Ng), encoded by the schizophrenia risk gene NRGN, is a calmodulin-binding protein enriched in the postsynaptic compartments, and its expression is reduced in the postmortem brains of patients with schizophrenia. Experience-dependent translation of Ng is critical for encoding contextual memory, and Ng regulates developmental plasticity in the primary visual cortex during the critical period. However, the overall impact of Ng on the neuronal signaling that regulates synaptic plasticity is unknown.
Slowpoke (Slo) potassium channels display extraordinarily high conductance, are synergistically activated by a positive transmembrane potential and high intracellular Ca2+ concentrations and are important targets for insecticides and antiparasitic drugs. However, it is unknown how these compounds modulate ion translocation and whether there are insect-specific binding pockets. Here, we report structures of Drosophila Slo in the Ca2+-bound and Ca2+-free form and in complex with the fungal neurotoxin verruculogen and the anthelmintic drug emodepside. Whereas the architecture and gating mechanism of Slo channels are conserved, potential insect-specific binding pockets exist. Verruculogen inhibits K+ transport by blocking the Ca2+-induced activation signal and precludes K+ from entering the selectivity filter. Emodepside decreases the conductance by suboptimal K+ coordination and uncouples ion gating from Ca2+ and voltage sensing. Our results expand the mechanistic understanding of Slo regulation and lay the foundation for the rational design of regulators of Slo and other voltage-gated ion channels.
In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A–E (1–5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1–5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1β1γ2 and α4β3γ2 receptors (IC50 1.5 and 1.0 μM, respectively). Although the structures of 1–6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.
Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state.
Machine learning is widely used in drug development to predict activity in biological assays based on chemical structure. However, the process of transitioning from one experimental setup to another for the same biological endpoint has not been extensively studied. In a retrospective study, we here explore different modeling strategies of how to combine data from the old and new assays when training conformal prediction models using data from hERG and NaV assays. We suggest to continuously monitor the validity and efficiency of models as more data is accumulated from the new assay and select a modeling strategy based on these metrics. In order to maximize the utility of data from the old assay, we propose a strategy that augments the proper training set of an inductive conformal predictor by adding data from the old assay but only having data from the new assay in the calibration set, which results in valid (well-calibrated) models with improved efficiency compared to other strategies. We study the results for varying sizes of new and old assays, allowing for discussion of different practical scenarios. We also conclude that our proposed assay transition strategy is more beneficial, and the value of data from the new assay is higher, for the harder case of regression compared to classification problems.
The SCN5A R1623Q mutation is one of the most common genetic variants associated with severe congenital long QT syndrome 3 (LQT3) in fetal and neonatal patients. To investigate the properties of the R1623Q mutation, we established an induced pluripotent stem cell (iPSC) cardiomyocyte (CM) model from a patient with LQTS harboring a heterozygous R1623Q mutation. The properties and pharmacological responses of iPSC-CMs were characterized using a multi-electrode array system. The biophysical characteristic analysis revealed that R1623Q increased open probability and persistent currents of sodium channel, indicating a gain-of-function mutation. In the pharmacological study, mexiletine shortened FPDcF in R1623Q-iPSC-CMs, which exhibited prolonged field potential duration corrected by Fridericia’s formula (FPDcF, analogous to QTcF). Meanwhile, E4031, a specific inhibitor of human ether-a-go-go-related gene (hERG) channel, significantly increased the frequency of arrhythmia-like early after depolarization (EAD) events. These characteristics partly reflect the patient phenotypes. To further analyze the effect of neonatal isoform, which is predominantly expressed in the fetal period, on the R1623Q mutant properties, we transfected adult form and neonatal isoform SCN5A of control and R1623Q mutant SCN5A genes to 293T cells. Whole-cell automated patch-clamp recordings revealed that R1623Q increased persistent Na+ currents, indicating a gain-of-function mutation. Our findings demonstrate the utility of LQT3-associated R1623Q mutation-harboring iPSC-CMs for assessing pharmacological responses to therapeutic drugs and improving treatment efficacy. Furthermore, developmental switching of neonatal/adult NaV1.5 isoforms may be involved in the pathological mechanisms underlying severe long QT syndrome in fetuses and neonates.
Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening.
Incorporation of noncanonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing human acid-sensing ion channel 1a (hASIC1a) variants in transiently transfected mammalian cells. We introduce 3 different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay, and live-cell crosslinking provides insight into the hASIC1a–psalmotoxin 1 (PcTx1) interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.
Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.
MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.
Protoxin II (ProTx II) is a high affinity gating modifier that is thought to selectively block the NaV1.7 voltage-dependent Na+ channel, a major therapeutic target for the control of pain. We aimed at producing ProTx II analogues entitled with novel functionalities for cell distribution studies and biochemical characterization of its NaV channel targets.
Gain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. Here it was tested whether any of the known human GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface—“super-trafficking”. Seven out of the 15 GOF mutants tested were seen to surface-traffic more efficiently than the wild type (WT) channel. Among these we found that levels of R231C KCNQ1 in the plasma membrane were 5-fold higher than the WT channel. This was shown to arise from the combined effects of enhanced efficiency of translocon-mediated membrane integration of the S4 voltage-sensor helix and from enhanced post-translational folding/trafficking that is related to energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 exhibits constitutive conductance, but also revealed that the single channel activity of this mutant is only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the effects of super-trafficking and constitutive channel activation, which together offset reduced channel activity. These investigations show that membrane protein super-trafficking can contribute to human disease.
Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons.
The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major CaVeat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.
The gene KCNT1 encodes the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2). Variants in the KCNT1 gene induce a gain-of-function (GoF) phenotype in ionic currents and cause a spectrum of intractable neurological disorders in infants and children, including epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Effective treatment options for KCNT1-related disease are absent, and novel therapies are urgently required. We describe the development of a novel class of oxadiazole KNa1.1 inhibitors, leading to the discovery of compound 31 that reduced seizures and interictal spikes in a mouse model of KCNT1 GoF.
The rising interest in KV7 modulators originates from their ability to evoke fundamental electrophysiological perturbations in a tissue-specific manner. A large number of therapeutic applications are, in part, based on the clinical experience with two broad-spectrum KV7 agonists, flupirtine and retigabine. Since precise molecular structures of human KV7 channel subtypes in closed and open states have only very recently started to emerge, computational studies have traditionally been used to analyze binding modes and direct the development of more potent and selective KV7 modulators with improved safety profiles. Herein, the synthetic and medicinal chemistry of small molecule modulators and the representative biological properties are summarized. Furthermore, new therapeutic applications supported by in vitro and in vivo assay data are suggested.
Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity.
Voltage-gated sodium channels (NaVs) are promising targets for analgesic and antiepileptic therapies. Although specificity between NaV subtypes may be desirable to target specific neural types, such as nociceptors in pain, many broadly acting NaV inhibitors are clinically beneficial in neuropathic pain and epilepsy. Here, we present the first systematic characterization of vixotrigine, a NaV blocker. Using recombinant systems, we find that vixotrigine potency is enhanced in a voltage- and use-dependent manner, consistent with a state-dependent block of NaVs. Furthermore, we find that vixotrigine potently inhibits sodium currents produced by both peripheral and central nervous system NaV subtypes, with use-dependent IC50 values between 1.76 and 5.12 μM. Compared with carbamazepine, vixotrigine shows higher potency and more profound state-dependent inhibition but a similar broad spectrum of action distinct from NaV1.7- and NaV1.8-specific blockers. We find that vixotrigine rapidly inhibits NaVs and prolongs recovery from the fast-inactivated state. In native rodent dorsal root ganglion sodium channels, we find that vixotrigine shifts steady-state inactivation curves. Based on these results, we conclude that vixotrigine is a broad-spectrum, state-dependent NaV blocker.
We applied a set of in silico and in vitro assays, compliant with the CiPA (Comprehensive In Vitro Proarrhythmia Assay) paradigm, to assess the risk of chloroquine or hydroxychloroquine‐mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin and azithromycin, drugs repurposed during the first wave of COVID‐19. Each drug or drug combination was tested in patch clamp assays on 7 cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay®) using control (healthy) or high‐risk cell populations, and in human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes. In each assay, concentration‐response curves encompassing and exceeding therapeutic free plasma levels were generated. Both chloroquine and hydroxychloroquine showed blocking activity against some potassium, sodium and calcium currents. Chloroquine and hydroxychloroquine inhibited IKr (IC50: 1µM and 3‐7 µM, respectively) and IK1 currents (IC50: 5 and 44 µM, respectively). When combining hydroxychloroquine with azithromycin, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50 > 300 ‐ 1000µM). Using Virtual Assay®, both antimalarials affected several TdP indicators, chloroquine being more potent than hydroxychloroquine. Effects were more pronounced in the high‐risk cell population. In hiPSC‐derived cardiomyocytes, all drugs showed early‐after‐depolarizations, except azithromycin. Combining chloroquine or hydroxychloroquine with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off‐label use in COVID‐19, chloroquine and hydroxychloroquine use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.
Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration–dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge–dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.
CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus (TRN) and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10,000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore, and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher’s exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modeled the effect of the biophysical properties of CaV3.3 channel variants on TRN excitability and found that compared with common variants, ultrarare CaV3.3 coding variants derived from control subjects significantly decreased TRN excitability (P = 0.011). When all rare variants were analyzed, there was a nonsignificant trend between variants that reduced TRN excitability and variants that either had no effect or increased TRN excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.
Ion channels are drug targets for neurologic, cardiac, and immunologic diseases. Many disease-associated mutations and drugs modulate voltage-gated ion channel activation and inactivation, suggesting that characterizing state-dependent effects of test compounds at an early stage of drug development can be of great benefit. Historically, the effects of compounds onion channel biophysical properties and voltage-dependent activation/inactivation could only be assessed using low-throughput, manual patch clamp recording techniques. In recent years, automated patch clamp (APC) platforms have drastically increased throughput. In contrast to their broad utilization in compound screening, APC platforms have rarely been used for mechanism of action studies, in large part due to the lack of sophisticated, scalable analysis methods to process the large amount of data generated by APC platforms. In the current study, we developed a highly efficient and scalable software workflow to overcome this challenge. This method, to our knowledge the first of its kind, enables automated curve fitting and complex analysis of compound effects. Using voltage-gated sodium channels as an example, we were able to immediately assess the effects of test compounds on a spectrum of biophysical properties, including peak current, voltage-dependent steady state activation/inactivation, and time constants of activation and fast inactivation. Overall, this automated data analysis method provides a novel solution for in-depth analysis of large-scale APC data, and thus will significantly impact ion channel research and drug discovery.
Malignant migrating partial seizures of infancy is a rare, devastating form of epilepsy most commonly associated with gain-of-function mutations in the potassium channel, Slack. Not only is this condition almost completely pharmacoresistant, there are not even selective drug-like tools available to evaluate whether inhibition of these overactivated, mutant Slack channels may represent a viable path forward toward new antiepileptic therapies. Therefore, we used a highthroughput thallium flux assay to screen a drug-like, 100 000-compound library in search of inhibitors of both wild-type and a disease-associated mutant Slack channel. Using this approach, we discovered VU0606170, a selective Slack channel inhibitor with low micromolar potency. Critically, VU0606170 also proved effective at significantly decreasing the firing rate in overexcited, spontaneously firing cortical neuron cultures. Taken together, our data provide compelling evidence that selective inhibition of Slack channel activity can be achieved with small molecules and that inhibition of Slack channel activity in neurons produces efficacy consistent with an antiepileptic effect. Thus, the identification of VU0606170 provides a much-needed tool for advancing our understanding of the role of the Slack channel in normal physiology and disease as well as its potential as a target for therapeutic intervention.
The novel coroNaVirus, SARS-CoV-2, has been identified as the causative agent for the current coroNaVirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.
There is an increasing expectation that computational approaches may supplement existing human decision-making. Frontloading of models for cardiac safety prediction is no exception to this trend, and ongoing regulatory initiatives propose use of high-throughput in vitro data combined with computational models for calculating proarrhythmic risk. Evaluation of these models requires robust assessment of the outcomes. Using FDA Adverse Event Reporting System reports and electronic healthcare claims data from the Truven-MarketScan US claims database, we quantify the incidence rate of arrhythmia in patients and how this changes depending on patient characteristics. First, we propose that such datasets are a complementary resource for determining relative drug risk and assessing the performance of cardiac safety models for regulatory use. Second, the results suggest important determinants for appropriate stratification of patients and evaluation of additional drug risk in prescribing and clinical support algorithms and for precision health.
In the recent years, the progress in genetic analysis and next-generation sequencing technologies have opened up exciting landscapes for diagnosis and study of molecular mechanisms, allowing the determination of a particular mutation for individual patients suffering from hereditary red blood cell-related diseases or anemia. However, the huge amount of data obtained makes the interpretation of the results and the identification of the pathogenetic variant responsible for the diseases sometime difficult. Moreover, there is increasing evidence that the same mutation can result in varying cellular properties and different symptoms of the disease. Even for the same patient, the phenotypic expression of the disorder can change over time. Therefore, on top of genetic analysis, there is a further request for functional tests that allow to confirm the pathogenicity of a molecular variant, possibly to predict prognosis and complications (e.g., vaso-occlusive pain crises or other thrombotic events) and, in the best case, to enable personalized theranostics (drug and/or dose) according to the disease state and progression. The mini-review will reflect recent and future directions in the development of diagnostic tools for red blood cell-related diseases and anemias. This includes point of care devices, new incarnations of well-known principles addressing physico-chemical properties, and interactions of red blood cells as well as high-tech screening equipment and mobile laboratories.
Malfunctions of voltage-gated sodium and calcium channels (SCN and CACNA1 genes) have been associated with severe neurologic, psychiatric, cardiac and other diseases. Altered channel activity is frequently grouped into gain or loss of ion channel function (GOF or LOF, respectively) which is not only corresponding to clinical disease manifestations, but also to differences in drug response. Experimental studies of channel function are therefore important, but laborious and usually focus only on a few variants at a time. Based on known gene-disease-mechanisms, we here infer LOF (518 variants) and GOF (309 variants) of likely pathogenic variants from disease phenotypes of variant carriers. We show regional clustering of inferred GOF and LOF variants, respectively, across the alignment of the entire gene family, suggesting shared pathomechanisms in the SCN/CACNA1 genes. By training a machine learning model on sequence- and structure-based features we predict LOF- or GOF- associated disease phenotypes (ROC = 0.85) of likely pathogenic missense variants. We then successfully validate the GOF versus LOF prediction on 87 functionally tested variants in SCN1/2/8A and CACNA1I (ROC = 0.73) and in exome-wide data from > 100.000 cases and controls. Ultimately, functional prediction of missense variants in clinically relevant genes will facilitate precision medicine in clinical practice.
Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) - not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances - not antagonizes - the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.
Acid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to synaptic plasticity, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding induces ASIC1a conformational changes that are different from those induced by protonation and likely represent a distinct closed state. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this CaVity as an important site for the development of ASIC-targeting therapeutics. Significance: Neuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way toward future analgesics.
The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries.
In this issue of SLAS Discovery, we present a special collection of manuscripts, including three original research papers and one review, that reflect recent advances and continuing challenges in the development and application of assay technologies to drug discovery for ion channel targets. First, though, we provide our perspectives on the specific challenges and opportunities in this field.
Rationale: Partial or complete loss of function variants in SCN5A are the most common genetic cause of the arrhythmia disorder Brugada Syndrome (BrS1). However, the pathogenicity of SCN5A variants is often unknown or disputed; 80% of the 1,390 SCN5A missense variants observed in at least one individual to date are variants of uncertain significance (VUS). The designation of VUS is a barrier to the use of sequence data in clinical care. Objective: We selected 83 variants for study: 10 previously studied control variants, 10 suspected benign variants, and 63 suspected Brugada Syndrome-associated variants, selected on the basis of their frequency in the general population and in patients with Brugada Syndrome. We used high-throughput automated patch clamping to study the function of the 83 variants, with the goal of reclassifying variants with functional data. Methods and Results: Ten previously studied variants had functional properties concordant with published manual patch clamp data. All 10 suspected benign variants had wildtype-like function. 22 suspected BrS variants had loss of channel function (10% normalized peak current) and 23 variants had partial loss of function (10-50% normalized peak current). The 73 previously unstudied variants were initially classified as likely benign (n=2), likely pathogenic (n=11), or VUS (n=60). After the patch clamp studies, 16 variants were benign/likely benign, 47 were pathogenic/likely pathogenic, and only 10 were still VUS. 8/22 loss of function variants were partially rescuable by incubation at lower temperature or pretreatment with a sodium channel blocker. Structural modeling identified likely mechanisms for loss of function including altered thermostability, and disruptions to alpha helices, disulfide bonds, or the permeation pore. Conclusions: High-throughput automated patch clamping enabled the reclassification of the majority of tested VUS’s in SCN5A.
GS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (INaL). Here, we took advantage of a throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (INaP) recorded from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We compared GS-967 and eleclazine to the antiarrhythmic drug lidocaine, the prototype INaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of INaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of INaP (IC50=0.07 and 0.6 μM, respectively) than ranolazine (7.8 μM), lidocaine (133.5 μM) and lacosamide (158.5 μM). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared to the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the significantly higher association rates (KON) and moderate unbinding rate (KOFF) of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine. Significance statement:We investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique features including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.
Background: KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease IKr can cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study. Objective: To report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2. Methods: We develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region. Results: For 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produce IKr, but that some variants which traffic normally may still be functionally compromised. Conclusions: Here, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in KV11.1 and to other cell surface proteins. Clinical Implications: Hundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.
Voltage-gated Na+ (NaV) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial NaV channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial NaV remains largely unexplored. Here we systematically screened 39 NaV modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human NaV1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both NaV1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases NaV1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of NaV1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian NaV channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.
The function of the voltage-gated KCNQ1 potassium channel is regulated by co-assembly with KCNE auxiliary subunits. KCNQ1-KCNE1 channels generate the slow delayed rectifier current, IKs, which contributes to the repolarization phase of the cardiac action potential. A three amino acid motif (F57-T58-L59, FTL) in KCNE1 is essential for slow activation of KCNQ1-KCNE1 channels. However, how this motif interacts with KCNQ1 to control its function is unknown. Combining computational modeling with electrophysiological studies, we developed structural models of the KCNQ1-KCNE1 complex that suggest how KCNE1 controls KCNQ1 activation. The FTL motif binds at a cleft between the voltage-sensing and pore domains and appears to affect the channel gate by an allosteric mechanism. Comparison with the KCNQ1-KCNE3 channel structure suggests a common transmembrane-binding mode for different KCNEs and illuminates how specific differences in the interaction of their triplet motifs determine the profound differences in KCNQ1 functional modulation by KCNE1 versus KCNE3.
Conventionally, manual patch-clamp electrophysiological approaches are the gold standard for studying ion channel function in neurons. However, these approaches are labor-intensive, yielding low-throughput results, and are therefore not amenable for compound profiling efforts during the early stages of drug discovery. The SyncroPatch 384PE has been successfully implemented for pharmacological experiments in heterologous overexpression systems that may not reproduce the function of voltage-gated ion channels in a native, heterogeneous environment. Here, we describe a protocol allowing the characterization of endogenous voltage-gated potassium (KV) and sodium (NaV) channel function in developing primary rat cortical cultures, allowing investigations at a significantly improved throughput compared with manual approaches. Key neuronal marker expression and microelectrode array recordings of electrophysiological activity over time correlated well with neuronal maturation. Gene expression data revealed high molecular diversity in KV and NaV subunit composition throughout development. Voltage-clamp experiments elicited three major current components composed of inward and outward conductances. Further pharmacological experiments confirmed the endogenous expression of functional KV and NaV channels in primary cortical neurons. The major advantages of this approach compared with conventional manual patch-clamp systems include unprecedented improvements in experimental ease and throughput for ion channel research in primary neurons. These efforts demonstrated feasibility for primary neuronal ion channel investigation with the SyncroPatch, providing the foundation for future studies characterizing biophysical changes in endogenous ion channels in primary systems associated with disease or development.
Mathematical models of ion channels, which constitute indispensable components of action potential models, are commonly constructed by fitting to whole-cell patch-clamp data. In a previous study, we fitted cell-specific models to hERG1a (KV11.1) recordings simultaneously measured using an automated high-throughput system, and studied cell-cell variability by inspecting the resulting model parameters. However, the origin of the observed variability was not identified. Here, we study the source of variability by constructing a model that describes not just ion current dynamics, but the entire voltage-clamp experiment. The experimental artefact components of the model include: series resistance, membrane and pipette capacitance, voltage offsets, imperfect compensations made by the amplifier for these phenomena, and leak current. In this model, variability in the observations can be explained by either cell properties, measurement artefacts, or both. Remarkably, by assuming that variability arises exclusively from measurement artefacts, it is possible to explain a larger amount of the observed variability than when assuming cell-specific ion current kinetics. This assumption also leads to a smaller number of model parameters. This result suggests that most of the observed variability in patch-clamp data measured under the same conditions is caused by experimental artefacts, and hence can be compensated for in post-processing by using our model for the patch-clamp experiment. This study has implications for the question of the extent to which cell-cell variability in ion channel kinetics exists, and opens up routes for better correction of artefacts in patch-clamp data.
In response to the ongoing shift of the regulatory cardiac safety paradigm, a recent White Paper proposed general principles for developing and implementing proarrhythmia risk prediction models. These principles included development strategies to validate models, and implementation strategies to ensure a model developed by one lab can be used by other labs in a consistent manner in the presence of lab-to-lab experimental variability. While the development strategies were illustrated through the validation of the model under the Comprehensive In vitro Proarrhythmia Assay (CiPA), the implementation strategies have not been adopted yet.
The scorpion toxin AmmTx3 is a specific blocker of KV4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.
Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide NaV1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of NaV1.7. Compound 24 showed a robust PK/PD response in a NaV1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.
Objective Pathogenic variants in KCNB1, encoding the voltage‐gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathy (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell‐surface expression.Methods We evaluated a series of 17 KCNB1 variants associated with DEE or other neurodevelopmental disorders (NDDs) to rapidly ascertain channel dysfunction using high‐throughput functional assays. Specifically, we investigated the biophysical properties and cell‐surface expression of variant KV2.1 channels expressed in heterologous cells using high‐throughput automated electrophysiology and immunocytochemistry–flow cytometry.Results Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage dependence of activation and/or inactivation, as homotetramers or when coexpressed with wild‐type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell‐surface expression. Interpretation Our study establishes a platform for rapid screening of KV2.1 functional defects caused by KCNB1 variants associated with DEE and other NDDs. This will aid in establishing KCNB1 variant pathogenicity and the mechanism of dysfunction, which will enable targeted strategies for therapeutic intervention based on molecular phenotype.
Voltage-gated sodium (NaV) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human NaV1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from NaV1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the NaV1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective NaV channel antagonists.
Predicting how pharmaceuticals may affect heart rhythm is a crucial step in drug-development, and requires a deep understanding of a compound’s action on ion channels. In vitro hERG-channel current recordings are an important step in evaluating the pro-arrhythmic potential of small molecules, and are now routinely performed using automated high-throughput patch clamp platforms. These machines can execute traditional voltage clamp protocols aimed at specific gating processes, but the array of protocols needed to fully characterise a current is typically too long to be applied in a single cell. Shorter high-information protocols have recently been introduced which have this capability, but they are not typically compatible with high-throughput platforms. We present a new high-information 15 s protocol to characterise hERG (KV11.1) kinetics, suitable for both manual and high-throughput systems. We demonstrate its use on the Nanion SyncroPatch 384PE, a 384 well automated patch clamp platform, by applying it to CHO cells stably expressing hERG1a. From these recordings we construct 124 cell-specific variants/parameterisations of a hERG model at 25 °C. A further 8 independent protocols are run in each cell, and are used to validate the model predictions. We then combine the experimental recordings using a hierarchical Bayesian model, which we use to quantify the uncertainty in the model parameters, and their variability from cell to cell, which we use to suggest reasons for the variability. This study demonstrates a robust method to measure and quantify uncertainty, and shows that it is possible and practical to use high-throughput systems to capture full hERG channel kinetics quantitatively and rapidly. Statement of Significance We present a method for high-throughput characterisation of hERG potassium channel kinetics, via fitting a mathematical model to results of over one hundred single cell patch clamp measurements collected simultaneously on an automated voltage clamp platform. The automated patch clamp data are used to parameterise a mathematical ion channel model fully, opening a new era of automated and rapid development of mathematical models from quick and cheap experiments. The method also allows ample data for independent validation of the models and enables us to study experimental variability and propose its origins. In future the method can be applied to characterise changes to hERG currents in different conditions, for instance at different temperatures (see Part II of the study) or under mutations or the action of pharmaceuticals; and should be easily adapted to study many other currents.
Ion channel behaviour can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing and inactivating) are commonly represented with Q10 coefficients or an Eyring relationship. In this paper we assess the validity of these representations by characterising channel kinetics at multiple temperatures. We focus on the hERG channel, which is important in drug safety assessment and commonly screened at room temperature, so that results require extrapolation to physiological temperature. In Part I of this study we established a reliable method for high-throughput characterisation of hERG1a (KV11.1) kinetics, using a 15 second information-rich optimised protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using CHO cells over-expressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch clamp platform, with temperature control. We characterise the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37 °C, and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients: it broadly follows a generalised, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol-dependent. Our results show that a direct fit using our 15 second protocol best represents hERG kinetics at any given temperature, and suggests that predictions from the Generalised Eyring theory may be preferentially used if no experimentally-derived data are available. Statement of Significance Ion channel currents are highly sensitive to temperature changes. Yet because many experiments are performed more easily at room temperature, it is common to extrapolate findings to physiological temperatures through the use of Q10 coefficients or Eyring rate theory. By applying short, information-rich protocols that we developed in Part I of this study we identify how kinetic parameters change over temperature. We find that the commonly-used Q10 and Eyring formulations are incapable of describing the parameters’ temperature dependence, a more Generalised Eyring relationship works well, but remeasuring kinetics and refitting a model is optimal. The findings have implications for the accuracy of the many applications of Q10 coefficients in electrophysiology, and suggest that care is needed to avoid misleading extrapolations in their many scientific and industrial pharmaceutical applications.
AimsCurrent treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by KV11.1 channels. Small molecule activators of KV11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of KV11.1 that impairs transition to the inactivated state, to restore function to heterozygous KV11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. Methods and results ICA-105574 effectively restored KV11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective KV11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The KV11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. Conclusions KV11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the CaVeat that there may be a risk of overcorrection that could itself be pro-arrhythmic.
Background: Voltage-gated potassium channel KV4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus, is a blocker of KV4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac KV4.2 channels remains unclear. Conclusion :AmmTx3 toxin can be chemically synthesized and used as a KV4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.
Pathogenic variants in KCNB1, encoding the voltage-gated potassium channel KV2.1, are associated with developmental and epileptic encephalopathies (DEE). Previous functional studies on a limited number of KCNB1 variants indicated a range of molecular mechanisms by which variants affect channel function, including loss of voltage sensitivity, loss of ion selectivity, and reduced cell-surface expression. We evaluated a series of 17 KCNB1 variants associated with DEE or neurodevelopmental disorder (NDD) to rapidly ascertain channel dysfunction using high-throughput functional assays. Specifically, we investigated the biophysical properties and cell-surface expression of variant KV2.1 channels expressed in heterologous cells using high-throughput automated electrophysiology and immunocytochemistry-flow cytometry. Pathogenic variants exhibited diverse functional defects, including altered current density and shifts in the voltage-dependence of activation and/or inactivation, as homotetramers or when co-expressed with wild-type KV2.1. Quantification of protein expression also identified variants with reduced total KV2.1 expression or deficient cell-surface expression. Our study establishes a platform for rapid screening of functional defects of KCNB1 variants associated with DEE and other NDDs, which will aid in establishing KCNB1 variant pathogenicity and may enable discovery of targeted strategies for therapeutic intervention based on molecular phenotype.
Background KCNH2 encodes the human ether-à-go-go-related gene (hERG) potassium channel, which passes the rapid delayed rectifier potassium current, IKr. Loss-of-function variants in KCNH2 cause long QT syndrome type 2 (LQTS2) which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants however are likely to be benign. Objective To develop a high-throughput assay for discriminating between pathogenic and benign KCNH2 variants. Methods Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 (HEK293) cell lines were phenotyped using an automated patch-clamp platform (SyncroPatch 384PE) and a cell surface ELISA assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In HEK293 using a bicistronic vector was performed using SyncroPatch 384PE. Results In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured by ELISA can be explained by changes in gating properties of the variant channels. Amongst 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of 13 pore-domain variants studied, 11 had a dominant-negative expression defect whilst the remaining two had enhanced inactivation gating resulting in a dominant-negative phenotype. Conclusions High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants
Objective: We identified a novel de novo KCNT1 variant in a patient with early‐infantile epileptic encephalopathy (EIEE) and status dystonicus, a life‐threatening movement disorder. We determined the functional consequences of this variant on the encoded KNa1.1 channel to investigate the molecular mechanisms responsible for this disorder. Methods: A retrospective case review of the proband is presented. We performed manual and automated electrophysiologic analyses of the KCNT1‐L437F variant expressed heterologously in Chinese hamster ovary (CHO) cells in the presence of channel activators/blockers. Results: The KCNT1‐L437F variant, identified in a patient with refractory EIEE and status dystonicus, confers a gain‐of‐function channel phenotype characterized by instantaneous, voltage‐dependent activation. Channel openers do not further increase L437F channel function, suggesting maximal activation, whereas channel blockers similarly block wild‐type and variant channels. We further demonstrated that KCNT1 current can be measured on a high‐throughput automated electrophysiology platform with potential value for future screening of novel and repurposed pharmacotherapies. Interpretation: A novel pathogenic variant in KCNT1 associated with early‐onset, medication‐refractory epilepsy and dystonia causes gain‐of‐function with rapid activation kinetics. Our findings extend the genotype–phenotype relationships of KCNT1 variants to include severe dystonia.
Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs’ actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. CaV1.2 or NaV1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.
The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.
Highlights: Development of six potent diazirine-containing photoprobes based on Huwentoxin-IV. Photoprobes specifically photolabel purified bacterial-NaV1.7 VSD2 chimeric channels. Proteomic mass spectrometry identifies binding site on S1-S2 loop and S3 helix. Proposed model of HwTx-IV binding reveals importance of K27 and R29. Summary: Voltage-gated sodium (NaV) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype NaV1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate NaV1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of NaV1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on NaV channels.
Background The voltage-gated K+-channel KV11.1 has a central role in cardiac repolarization. Blockage of KV11.1 has been linked to severe cardiovascular side effects, such as acquired long QT syndrome (aLQTS), torsade de pointes arrhythmia and sudden cardiac death (SCD). KV11.1 is susceptible to unspecific drug interactions due to the presence of two aromatic amino acids residing in the inner vestibule of the pore. These aromatic residues are also present in the equine orthologue of KV11.1. This suggests that equine KV11.1 may also be prone to high-affinity block by a range of different chemical entities, which potentially could cause severe cardiac side effects and SCD in horses. Aim To screen a series of commonly used drugs in equine medicine for interaction with KV11.1. Methods High-throughput screening of selected compounds on human KV11.1 expressed in a mammalian cell line was performed using an automated patch clamp system, the SyncroPatch 384PE (Nanion Technologies, Munich, Germany). Results were validated on equine KV11.1 expressed in CHO-K1 cells by manual patch clamp. Results Acepromazine maleat (IC50 = 0.5 μM) trimethoprim (IC50 = 100 μM), diphenhydramine hydrochloride (IC50 = 2 μM) and cyproheptadine hydrochloride (IC50 = 1.84 μM) inhibited equine KV11.1 current at clinically relevant drug concentrations. Conclusion The results suggest that drug interaction with KV11.1 can occur in horses and that some drugs potentially may induce repolarization disorders in horses.
Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (NaV) channel NaV1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human NaV1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural NaV1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate NaV1.7 involvement in cellular excitability and pain.
Piezo1 is a mechanosensitive ion channel that is believed to be expressed in red blood cells (RBCs), mainly supported by the findings that mutations of PIEZO1 gene are associated with the RBC disease Hereditary Xerocytosis. So far several mutations have been reported, e.g. R2456H, T2127M and E2496ELE, to exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type. However, characterisation of the mutated ion channel has almost exclusively been performed based on heterologous expression in cell lines and recordings in RBCs were rather of episodic character. Here we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp based high-throughput screening assay for Piezo1 activity. It is the first electrophysiologic single-cell based screening ever performed on RBCs, demonstrating the Piezo1 gain-of-function mutation directly on RBCs. Thus we provide a putative routine approach for detecting functional (Piezo1) channel mutations as the molecular cause of rare anaemia that can become a standard method in specialised haematological centres.
The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.
Integral to the cell surface is channels, pumps, and exchanger proteins that facilitate the movement of ions across the membrane. Ion channels facilitate the passive movement of ions down an electrochemical gradient. Ion pumps actively use energy to actively translocate ions, often against concentration or voltage gradients, while ion exchangers utilize energy to couple the transport of different ion species such that one ion moves down its gradient and the released free energy is used to drive the movement of a different ion against its electrochemical gradient. Some ion pumps and exchangers may be electrogenic, i.e., the ion transport they support is not electrically neutral and generates a current. Functions of these pore-forming membrane proteins include the establishment of membrane potentials, gating of ions flows across the cell membrane to elicit action potentials and other electrical signals, as well as the regulation of cell volumes. The major forms of ion channels include voltage-, ligand-, and signal-gated channels. In this review, we describe mammalian voltage dependent Na (NaV) channels.
Sweating is a fundamental process required for human thermoregulation. In today’s modern society, however, extensive sweating is rather considered unpleasant or embarrassing, or can even cause severe psychosocial pressure. Sweat reduction by antiperspirants is therefore of huge cosmetic interest. Currently, the global use of aluminum salts as antiperspirants is controversial, but no alternatives exist so far. We developed a new concept for sweat reduction which is based on directly targeting primary fluid secretion in human sweat glands. We identified a long searched for key player in human sweat glands - the ion channel TMEM16A, also known as ANO1. We extensively characterized TMEM16A and its function in native human sweat glands and sweat gland tissue culture cells by using a wide variety of different techniques such as immunohistological staining, chloride flux assays, automated patch clamping as well as state-of-the-art CRISPR/ Cas9 genome editing technology. We generated a proprietary cell-based assay to emulate TMEM16A function in a cellular sweat gland environment. We combined this cell-based assay with our cherry-picked compound libraries and performed high-throughput screening campaigns which uncovered smallmolecule modulators of TMEM16A. In silico and in vitro toxicological assessments as well as stability and formulation tests were performed and yielded compounds that are currently being tested for their sweat reduction efficacy in vivo.
Background: SCN5A mutations can lead to different cardiac diseases. Recently, SCN5A mutations have been linked to the clinical entity multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by ventricular ectopy and dilated cardiomyopathy. Methods & Results: A family with a uniform MEPPC-like phenotype, associated with complex atrial and ventricular arrhythmias and dilated cardiomyopathy caused by a high frequency of ventricular ectopy was clinically assessed. Screening of the SCN5A gene revealed a missense mutation in the linker between segments 3 and 4 in domain 1 of the NaV1.5 protein, resulting in a glycine to aspartate substitution at position 213 (G213D). The phenotype co-segregated with the missense mutation. Electrophysiological studies of wild type (WT) hNaV1.5 and hNaV1.5_G213D expressed in CHO-K cells showed that the voltage of half-maximal activation (V½) was significantly more negative for hNaV1.5_G213D compared to WT (V½ = −38.7 ± 0.5 mV for WT and V½ = −42.4 ± 0.5 mV for G213D; P 0.001). This suggests activation of NaV1.5_G231D at more negative potentials. The V½ of steady-state inactivation was significantly shifted towards more positive values for NaV1.5_G213D (V½ = −86.7 ± 0.2 mV for WT and −82.2 ± 0.3 mV for G213D; P 0.001), also contributing to a gain-of-function phenotype. Flecainide and amiodarone markedly reduced premature atrial and ventricular contractions in four patients. Conclusion: The NaV1.5_G213D mutation is associated with a gain-of-function phenotype, multifocal atrial and ventricular ectopy and dilated cardiomyopathy. Since patients with a MEPPC-like phenotype may specifically benefit from Class-1 antiarrhythmic drugs or amiodarone, clinical identification of this disease entity is important. Note: Electrophysiological analysis of heteromers (NaV1.5 and NaV1.5_G213D mutation) were executed on the SyncroPatch 384PE. Data are not shown in the publication.
Many ion channels, including NaV1.7, CaV1.3, and KV1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the NaV1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered NaV1.7-selective chemical scaffolds. Hence, we address a major hurdle in NaV1.7 drug discovery, and this mechanistic approach to assay design is applicable to CaV3.1, KV1.3, and many other ion channels to facilitate drug discovery.
Background: The explosive growth in known human gene variation presents enormous challenges to current approaches for variant classification that have implications for diagnosis and treatment of many genetic diseases. For disorders caused by mutations in cardiac ion channels as in congenital arrhythmia syndromes, in vitro electrophysiological evidence has high value in discriminating pathogenic from benign variants, but these data are often lacking because assays are cost, time, and labor intensive. Methods: We implemented a strategy for performing high-throughput functional evaluations of ion channel variants that repurposed an automated electrophysiological recording platform developed previously for drug discovery. Results: We demonstrated the success of this approach by evaluating 78 variants in KCNQ1, a major gene involved in genetic disorders of cardiac arrhythmia susceptibility. We benchmarked our results with traditional electrophysiological approaches and observed a high level of concordance. This strategy also enabled studies of dominant-negative behavior of variants exhibiting severe loss-of-function. Overall, our results provided functional data useful for reclassifying >65% of the studied KCNQ1 variants. Conclusions: Our results illustrate an efficient and high-throughput paradigm linking genotype to function for a human cardiac ion channel that will enable data-driven classification of large numbers of variants and create new opportunities for precision medicine.
Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA—a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.
Voltage-gated KV1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of KV1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether KV1.3 is required for their induction and function is unclear. Here we show, using KV1.3-deficient rats, that KV1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in KV1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of KV1.3 under these specific settings. However, experiments with KV1.3 KO rats and KV1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both KV1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on KV1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.
Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel NaV1.7. By testing a group of NaV1.7 reference compounds’ IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z’ factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel KV1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.
GS-458967, 6-(4-(Trifluoromethoxy)phenyl)-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (GS967) is a recently described, novel, sodium channel inhibitor exhibiting potent antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current. However, there has been no reported systematic investigation of the effects of this compound on isolated sodium channels. Here, we examined the effects of GS967 on peak (INaP) and late (INaL) sodium current recorded from cells that heterologously expressed human cardiac voltage-gated sodium channel, the principle cardiac sodium channel. As previously described, we observed that GS967 exerted tonic block of INaL (63%) to a significantly greater extent than INaP (19%). However, GS967 also caused a reduction of INaP in a frequency-dependent manner, consistent with use-dependent block (UDB). GS967 evoked more potent UDB of INaP (IC50 = 0.07 µM) than ranolazine (16 µM) and lidocaine (17 µM). Use-dependent block was best explained by a significant slowing of recovery from fast and slow inactivation with a significant enhancement of slow inactivation in the presence of GS967. Furthermore, GS967 was found to exert these same effects on a prototypical long QT syndrome mutation (delKPQ). An engineered mutation at an interaction site for local anesthetic agents (F1760A) partially attenuated the effect of GS967 on UDB, but had no effect on tonic INaL block. We conclude that GS967 is a preferential inhibitor of INaL, but it also exerts previously unreported strong effects on slow inactivation and recovery from inactivation, resulting in substantial UDB that is not entirely dependent on a known interaction site for local anesthetic agents.
Objective: To perform functional characterization of a potentially pathogenic KCNB1 variant identified by clinical exome sequencing of a proband with a neurodevelopmental disorder that included epilepsy and centrotemporal spikes on EEG. Methods: Whole-exome sequencing identified the KCNB1 variant c.595A.T (p.Ile199Phe). Biochemical and electrophysiologic experiments were performed to determine whether this variant affected protein expression, trafficking, and channel functional properties. Results: Biochemical characterization of the variant suggested normal protein expression and trafficking. Functional characterization revealed biophysical channel defects in assembled homotetrameric and heterotetrameric channels. Conclusions: The identification of the KCNB1 variant c.595A.T (p.Ile199Phe) in a neurodevelopmental disorder that included epilepsy with centrotemporal spikes expands the phenotypic spectrum of epilepsies associated with KCNB1 variants. The KCNB1-I199F variant exhibited partial loss of function relative to the wild-type channel. This defect is arguably less severe than previously reported KCNB1 variants, suggesting the possibility that the degree of KCNB1 protein dysfunction may influence disease severity.
We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration–response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of 50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell–derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and new therapeutic targets are urgently needed. One of the hallmarks of cancer is changed pH-homeostasis and potentially pH-sensors may play an important role in cancer cell behavior. Two-pore potassium channels (K2P) are pH-regulated channels that conduct a background K+ current, which is involved in setting the plasma membrane potential (Vm). Some members of the K2P superfamily were reported as crucial players in driving tumor progression. The aim of this study was to investigate pH-regulated K+ currents in PDAC cells and determine possible effects on their pathological phenotype. Using a planar high-throughput patch-clamp system (SyncroPatch 384PE) we identified a pH-regulated K+ current in the PDAC cell line BxPC-3. The current was inhibited by extracellular acidification and intracellular alkalization. Exposure to a set of different K+ channel inhibitors, and the TREK-1 (K2P2.1)–specific activator BL1249, TREK-1 was identified as the main component of pH-regulated current. A voltage-sensor dye (VF2.1.Cl) was used to monitor effects of pH and BL1249 on Vm in more physiological conditions and TREK-1–mediated current was found as critical player in setting Vm. We assessed a possible role of TREK-1 in PDAC progression using cell proliferation and migration assays and observed similar trends with attenuated proliferation/migration rates in acidic (pH 7.0) and alkaline (pH > 7.4) conditions. Notably, BL1249 inhibited both PDAC cell proliferation and migration indicating that hyperpolarization of Vm attenuates cancer cell behavior. TREK-1 may therefore be a promising novel target for PDAC therapy.
Introduction From a drug discovery point of view, ion channels are very interesting and challenging targets. Over the past decade, great efforts have been made in developing platforms for patch clamp-based high-quality screening of ion channels in discovering new drug candidates as well for evaluating their safety profiles. Indeed, the automated patch clamp (APC) has recently reached the data throughput requirements of high-throughput screening (HTS) allowing for new screening strategies with ion channel active compounds. Areas covered This editorial article comments on the past and present developments of APC-based drug screening. Furthermore, it also looks at the implications of APC technology meeting HTS-standards as well as its use in compound safety evaluation. Expert opinion In the imminent future, we will see a paradigm shift in ion channel drug screening toward using APC-based platforms for primary drug library screens. This way, the redundancy of the drug discovery process and the risk of false-negatives could be drastically reduced. Furthermore, cardiac safety can be addressed early, avoiding late-phase withdrawals with promising drug candidates. It is our firm belief that APC-based ion channel HTS will facilitate the discovery of candidates, which otherwise would have not been found, and shorten the drug development cycle, saving time and cost.
Background An in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology. Methods In this study, high-throughput electrophysiological recordings of human embryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go related gene (hERG) were performed utilizing an automated 384-well-patch-clamp system, which records up to 384 cells simultaneously. hERG channel inhibition, which is closely related to a drug-induced QT prolongation and is increasing the risk of sudden cardiac death, was investigated in the high-throughput screening patch-clamp system. Results In the automated patch-clamp measurements performed here, KV currents were investigated with high efficiency. Various hERG channel blockers showed concentration-dependent inhibition, the 50 % inhibitory concentrations (IC50) of those blockers were in good agreement with previous reports. Conclusions The high-throughput patch-clamp system has a high potential in the field of pharmacology, toxicology, and cardiac physiology, and will contribute to the acceleration of pharmaceutical drug development and drug safety testing.
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.
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.
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.
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.
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 . 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 ), 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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