• Nanion Technologies: Smart Tools for Ion Channel Research

    Nanion Technologies: Smart Tools for Ion Channel Research

  • SyncroPatch 384i: HTS Automated Patch Clamp

    SyncroPatch 384i: HTS Automated Patch Clamp

  • SURFE²R 96SE: Label-free HTS Transporter Screening

    SURFE²R 96SE: Label-free HTS Transporter Screening

  • Dynamic Clamp: Patchliner

    Dynamic Clamp: Patchliner

  • Bilayer recordings: Orbit product family

    Bilayer recordings: Orbit product family

  • CardioExcyte 96 SOL: Pacing Cardiomyocytes with Light

    CardioExcyte 96 SOL: Pacing Cardiomyocytes with Light

Our Product Portfolio

SyncroPatch 384

SyncroPatch 384

Patchliner

Patchliner

Port-a-Patch

Port-a-Patch

Port-a-Patch mini

Port-a-Patch mini

CardioExcyte 96

CardioExcyte 96

FLEXcyte 96

FLEXcyte 96

SURFE²R 96SE

SURFE²R 96SE

SURFE²R N1

SURFE²R N1

Orbit 16 TC

Orbit 16 TC

Orbit Mini

Orbit Mini

Vesicle Prep Pro

Vesicle Prep Pro

Buffer Solution

Buffer Solution

Ion Channels and Respiratory Diseases

Picture17


Did You Know: Ion Channels and their function in respiratory disease targets

The SARS-CoV-2 pandemic which emerged in late 2019, coupled with continued seasonal and pandemic influenza infections, has caused a significant problem for global public health and health systems worldwide. Antivirals and vaccines effective against these viruses are desperately needed in order to stem the pandemic and save lives.

A number of viroporins and ion channels are involved in viral infections and respiratory disorders, a selection is provided below.

Viroporins

Viroporins are small, hydrophobic integral membrane ion channels essential in the life cycle of a diverse range of RNA and DNA viruses. They perform multiple functions in the virus life cycle including viral entry, particle production and virus spread.

Viroporins are potential drug targets for block of viral replication and the spread of infection.


Useful Reviews
Scott, C., & Griffin, S. 2015. Viroporins: structure, function and potential as antiviral targets. Journal of General Virology

J Gen Virol

Nieva, J.L., Madan, V., Carrasco, L. 2012. Viroporins: structure and biological functions. Nature Reviews Microbiology.

Nat Microbiol

Wang, K., Xie, S., Sun, B. 2011. Viral proteins function as ion channels. Biochim Biophys Acta Biomembr.

Biochim_Biophys_Acta


Viroporins of Sars-CoV

Sars-CoV 3a forms a potassium channel located on the surface of Sars-CoV infected cells and promotes virus release (Lu, W., et al., 2006). Additionally, the Sars-CoV 3a and E proteins have been shown to activate the NLRP3 inflammasome (Chen, I-Y., et al., 2019) and are involved in virus replication and pathogenesis (Castaño-Rodriguez et al, 2018).


Viroporins of Influenza virus

The M2 protein of influenza A is a proton-gated, proton selective ion channel that is essential for viral replication (Jalily, P.H., et al, 2020). Amantadine is a well-known blocker of the influenza A M2 proton channel, and has been used as an anti-influenza drug for many years. Unfortunately it is associated with a number of severe side effects and is no longer recommended for the treatment of influenza A due to resistance as a result of mutations in the M2 protein


Measuring viroporins

Viroporins can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Ca2+-release activated calcium channel (CRAC)

Ca2+-release activated calcium channel (CRAC) is composed of Orai1, the pore forming subunit, and STIM1, the endoplasmic reticulum Ca2+ sensor. CRAC channels open in response to a depletion of intraluminal Ca2+ in the ER, allowing an influx of Ca2+ via a process known as store-operated calcium entry (SOCE). There is some evidence that CRAC channels inhibitors could be novel therapeutics for autoimmune of inflammatory conditions. The CRAC channel blocker, CM4620-IE, from CalciMedica has recently entered Phase II clinical trials to prevent a cytokine storm in COVID-19 patients.


Useful Reviews
Staudermann, T.A. 2018. CRAC channels as targets for drug discovery and development. Cell Calcium

Cell Calcium


Measuring CRAC Channels

CRAC channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Volume-regulated anion channels (VRAC), LRRC8

Volume-regulated anion channels (VRAC) are widely expressed in mammalian cells. They transport chloride ions and other organic ions, e.g. taurine or glutamate and maybe even water, across the plasma membrane VRAC channels are composed of LRRC8 heteromers of which there are 5 known variations, LRRC8A-E. Cell-to-cell cGAMP transmission via LRRC8/VRAC channels has been shown to play a crucial role in anti-viral immunity.

Key Publications and Reviews

Zhou, C., Chen, X., Planells-Cases, R., et al. 2020. Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity. Immunity

Immunity


Strange, K., Yamada, T., & Denton, G. 2019. A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel. Journal of General Physiology

J. Gen. Physiol.


Measuring VRAC Channels

VRAC Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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VRAC Publications using Nanion Instruments

Two pore channels (TPC)

Two pore channels (TPC) are calcium channels located on cellular endosomes. Activated by the second messenger NAADP and the phospholipid Phosphatidylinositol 3,5-bisphosphate, PtdIns(3,5)P2) the pore conducts primarily Ca2+ and Na+. It has been shown that they play a key role in Ebola virus infection and may provide a therapeutic target as an antiviral for Ebola virus and other filoviruses. In endosomal membranes they are responsible for entry of Ebola virus.

Key Publications

Sakurai, Y., Kolokoltsov, A.A., Chen, C-C., et al. 2015. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. Science

Science


Castonguay, J., Orth, J.H.C., Müller, T., et al. 2017. The two-pore channel TPC1 is required for efcient protein processing through early and recycling endosomes. Nature Scientific Reports

Science


Measuring Two Pore Channels

Two Pore Channels can be measured using automated patch clamp when expressed in cell lines, in lysosomes, or using the Orbit instruments when they are recombinantly expressed in bilayers.

Port-a-Patch
Automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Publications using Instruments from Nanion

Transporters - SLC6A19 in SARS-CoV-2

The sodium-dependent neutral amino acid transporter B0AT1 is a protein that in humans is encoded by the SLC6A19 gene. It mediates epithelial resorption of neutral amino acids across the apical membrane in the kidney and intestine. Recently it has been shown that the angiotensin-converting enzyme 2 (ACE2) and B0AT1 protein forms a complex for recognition of the SARS-CoV-2 virus, thereby playing a role in coronavirus recognition and infection.

Key Publications

Yan, R., Zhang, Y., Li, Y., et al. 2020. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science

Science


Measuring SLC19A and other Transporters

SLC19A can be measured using the SSM-electrophysiology technique. It may also be measured in bilayers if reconstituted and the effect of drugs acting on transporters can be measured using impedance.

SURFE2R
Transporter measurements

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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TMEM16A

TMEM16A is a calcium-activated chloride channel expressed in surface epithelium of the airway as well as submucosal glands and goblet cells. It is upregulated during inflammatory conditions. Recently a postitive modulator of TMEM16A has been shown to enhance anion and fluid secretion from cystic fibrosis patients. This type of approach presents a novel approach tot he treatment of cystic fibrosis and other respiratory disorders.

Key Publication

Danahay, H.L., Lilley, S., Fox, R., et al. 2019. TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis. American Journal of Respiratory and Critical Care Medicine

AJRCCM


Measuring TMEM16A

TMEM16A Channels can be measured using automated patch clamp when expressed in cell lines, and can be activated using the internal perfusion feature of the SyncroPatch 384i or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Application Notes

P2X3 in chronic cough

P2X receptors are trimeric ligand-gated ion channels which are activated by extracellular ATP. When activated, small monovalent cations pass through the pore. P2X chanenls have been shown to play a role in inflammatory, visceral, and neuropathic pain states, and in addition to this, in airways hyperreactivity, migraine, itch, and cancer pain. More recently, the antagonist of P2X2/3 and P2X3 receptors, MK‐7264 (gefapixant), has entered Phase III clinical trials for refractory and unexplained chronic cough.

Key Publications

Ford, A.P. & Undem, B.J. 2013. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders. Frontiers in Cellular Neuroscience

Frontiers in Cellular Neuroscience


Ford, A.P. 2012. In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization. Purinergic Signalling

Purinergic Signalling


Measuring P2X3 Channels

P2X3 Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

CardioExcyte 96
Cytotoxicity measurements

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Application Notes
Publications using Instruments from Nanion

CFTR

Cystic fibrosis (CF) is a fatal hereditary lung disease. Mutations in the cystic fibrosis conductance regulator (CFTR) channel have been shown to cause CF. CFTR is a regulated epithelial chloride channel and mutations cause a reduction in activity of the channel via a variety of mechanisms. CFTR dysfunction causes chronic mucus obstruction, neutrophilic inflammation and bacterial infection in CF airways. 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 and other respiratory disorders, such as chronic obstructive pulmonary disease (COPD) or asthma.

Useful Reviews

Noone, P.G. & Knowles, M.R. 2001. 'CFTR-opathies': disease phenotypes associated with cystic fibrosis transmembrane regulator gene mutations. Respiratory Research

Respiratory Research


Mall, M.A., & Hartl, D. 2014. CFTR: cystic fibrosis and beyond. European Respiratory Journal

Respiratory research


Measuring CFTR Channels

CFTR Channels can be measured using automated patch clamp when expressed in cell lines, or using the Orbit instruments when they are recombinantly expressed in bilayers. The CFTR channel is an ABC transporter and can also be measured using the SSM-electrophysiology technique.

SyncroPatch 384i
High throughput automated patch clamp

Orbit 16
Parallel bilayer recordings

SURFE2R
Transporter measurements

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Application Notes
Publications using Instruments from Nanion


CiPA - Comprehensive in vitro Proarrhythmia Assay

The Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative aims to replace the preclinical hERG current assay required under the ICH S7B safety pharmacology guidelines and clinical TQT study, which provides a surrogate marker of Proarrhythmia, with more translationally relevant assessments of proarrhythmic risk (Sager et al., 2014). CiPA intend to achieve this by evaluating proarrhythmic risk of evolving drug candidates based on an understanding of the electrophysiologic mechanisms responsible for proarrhythmia linked to Torsades de pointes (TdP) and QT prolongation.

The Q&As draft as endorsed August 2020 describe how nonclinical assays can be used as a part of an integrated risk assessment prior to first-in-human studies as they are used today, and in later stages of clinical development as a part of a combined nonclinical-clinical integrated risk assessment. If implemented, they can lead to a reduction in the number of ‘Thorough QT’ clinical studies and improved decision making at the time of a marketing application.

A webinar (Oct 15 and 16, 2020) will cover the background, motivation for, and overview of the new Q&As for ICH E14/S7B, followed by presentations on each of the main Q&A topics:
ICH E14 and S7B: Webinar on the recently released Draft Q&As


The CiPA workstreams

CiPA Work Groups

The Comprehensive in Vitro Proarrhythmia Assay (CiPA) paradigm was initiated in 2013. The Steering Team is comprised of partners from the US FDA, HESI, CSRC, SPS, Japan NIHS, PMDA, EMA and Health Canada. A number of participating organizations, amongst them Nanion Technologies, build the backbone of the initiative. The CiPA work streams include: In Silico, Myocyte, Ion Channel, and Clinical Translation working groups. 

Nanion is a committee member of the Ion Channel HTS Team and a participant in the Myocyte Working Group. Nanion has a long-standing interest and extensive experience in automated patch clamp screening of cardiac ion channels. Label-free contractility and extracellular field potential recordings of stem cell-derived cardiomyocytes (iPSC-CMs) is also available in our portfolio. Our instruments are used for safety screening by major pharmaceutical companies and CROs worldwide and we are happy to assist you in setting up your CiPA assays.


Results of Phase I Studies

CiPA Table Phase I smallThe Ion Channel Work Group finalized its phase I study in 2017. Besides further external sites, Nanion Technologies in Germany, USA and Japan participated with the Patchliner and the SyncroPatch 384PE in this study. 

The image on the left hand side displays the results of the blocking effect of 12 compounds on hERG. 
The results are in good agreement with manual patch clamp data (Crumb et al., 2016).  

 


Nanion's products in CiPA workstreams


icon sp96   SyncroPatch 384i - Patch clamp meets HTS 

Nanion CiPA IconThe SyncroPatch 384i is a high throughput patch clamp instrument recording from up to 384 cells simultaneously. The SyncroPatch 384i is the highest throughput patch clamp instrument on the market with giga-seal data quality. The SyncroPatch 384/768PE and the new system SyncroPatch 384i are used for CiPA ion channel working group studies.
Request the CiPA assay protocol for the SyncroPatch 384i here.

icon pl   Patchliner - Versatile and sophisticated

Nanion CiPA IconThe Patchliner is a fully automated planar patch clamp instrument recording from up to 8 cells simultaneously. With its vast experimental freedom and gigaseal data quality, the Patchliner is one of the most versatile patch clamp instruments on the market.
The Patchliner is used for CiPA ion channel working group studies.
Request the CiPA assay protocol for the Patchliner here

Icon CE   CardioExcyte 96 - Combined contractility, electrophysiology and cell viability

Nanion CiPA IconThe CardioExcyte 96 is a hybrid system recording both contractility and electrophysiology of intact cardiomyocyte networks. In addition, the base impedance is continuously and automatically monitored as a measure of acute and chronic cell activity. No subtleties of cytotoxic responses are missed, this includes non-contractile cell types such as hepatocyte-like cells or cancer cells as well as contractile cardiac cells. Impedance and extracellular field potential measurements are performed at high resolution, are non-invasive and label-free. The CardioExcyte 96 is a fully automated device, recording from 96 wells at a time. 
The CardioExcyte 96 is used for CiPA myocyte working group studies.
Request the CiPA assay protocol for the CardioExcyte 96 here.

Ion Channel Work Group (ICWG)

ICWG: Description and History

"The Ion Channel Working Group (ICWG), sponsored by the Safety Pharmacology Society (SPS), was established in December 2013. Its primary role is to work in close collaboration with the In Silico Working Group (ISWG) in providing ion channel support for the development of a computer model of the adult human ventricular myocyte, to be used as part of the CiPA initiative in predicting the clinical risk of drug-induced TdP."

"The original remit of the ICWG was: 1) to select key cardiac ion channels to include in the CiPA evaluation; 2) to develop robust, reliable and reproducible voltage clamp protocols required to generate data allowing the training and validation of the in silico model; 3) to define which biophysical and/or pharmacological properties of the channels to study for drug effects in order to optimize the predictivity of the model (e.g., potency (IC50), kinetic of block, rate/use/voltage dependence) and; 4) to define the requirements needed to transition the various ion channel protocols from manual to automated high throughput (HT) patch clamp platforms, in order to adapt to the screening environment present in most pharmaceutical companies."

Selected recombinant human channels: "IKr (hERG), ICa (L-type; Cav1.2), INa (Nav1.5 peak and late current); ITO (Kv4.3); IKs (KCNQ1 + KCNE1), and IK1 (Kir2.1). Following the selection, the ICWG set out to design standardized voltage clamp protocols for each of these channels."

(Source: Journal of Pharmacological and Toxicological Methods "The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative — Update on progress. Colatzky et al. (2016) ")


Progress of the Ion Channel Work Group

"Protocols were developed and manual experimental work was completed. The High-Throughput Systems (HTS) studies to evaluate and characterize the automated patch clamp systems is underway. The training compounds have been completed for both manual and automated systems and data analysis initiated. The second phase of the HTS study with the validation compounds is underway. Additional ion channel studies will likely be planned beyond 2017 to further validate the model."

CiPA Ion Channel Workstream

(Source: CiPAproject webpage "CiPA Workstream Timelines")


Data, Applications and Publications on the six Cardiac Ion Channels investigated by the "CiPA Ion Channel Work Group"

Myocyte Work Group (MWG)

 

MWG: Description and History

The role of human cardiac stem cell derived cardiomyocytes (hSC-CMs) within the CiPA paradigm is to confirm in silico reconstructions of the electrophysiologic effects of drugs. This will be accomplished by evaluating the acute effects of drugs on the electrical activity of hSC-CM's using higher throughput approaches that enable more comprehensive and robust assessments earlier in the drug discovery process. Typical techniques involved include assessing changes in the extracellular field potentials of spontaneously active hSC-CMs using multielectrode array (MEA) platforms, or changes in the action potential configuration recorded optically using voltage-sensing dyes (VSD)."

"A pilot study was conducted by the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee Myocyte Subteam to evaluate reproducibility and variability of electrophysiologic responses across cells, platforms and volunteer study sites for 8 compounds, Mexiletine, Nifedipine, E-4031, JNJ-303, Flecanide, Moxifloxacin, Quinidine and Ranolazine. Overall, most sites detected changes in repolarization consistent with clinical findings, with the greatest source of variability attributed to study site."

"More recently, the HESI Myocyte Subteam was awarded a Broad Area Announcement Grant from the FDA to extend the pilot study to a larger set of 28 compounds categorized according to high, intermediate, and low/no risk of proarrhythmia based on clinical findings. The effects of these CiPA reference standards will be evaluated by a core group in this Phase II Validation study across four MEA and two VSO technology platforms using two commercially viable hSC-CM preparations." Results will be compared with prospective in silico reconstructions (based on voltage clamp studies) and published studies employing hSC-CMs from non-core laboratories and consortia for the same 28 compounds."

(Source: Journal of Pharmacological and Toxicological Methods "The Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative — Update on progress. Colatzky et al. (2016) ")


Progress of the Myocyte Work Group

The publication detailing the results of both the Pilot and Validation Studies are available online. The review on best practices is available here.

CiPA Myocyte Workstream 2

(Source: CiPAproject webpage "CiPA Workstream Timelines")


Nanion for CiPA:

Data and Applications

Application Notes

Publications

Posters

Oral Presentations

Instrument Product Sheets

 

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