scholarly journals Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia

2017 ◽  
Vol 175 (12) ◽  
pp. 2312-2321 ◽  
Author(s):  
Damian C Bell ◽  
Mark L Dallas
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology

scholarly journals A Yoda1-Based Approach to Investigate Piezo1 Channels in Red Blood Cells Using Automated Patch Clamp Technology

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1031-1031
Author(s):  
Maria Giustina Rotordam ◽  
Elisa Fermo ◽  
Nadine Becker ◽  
Wilma Barcellini ◽  
Andrea Brüggemann ◽  
...  
Keyword(s):  
Shear Stress ◽  
Patch Clamp ◽  
Ion Channel ◽  
Red Blood Cells ◽  
High Throughput ◽  
Blood Cells ◽  
Screening Assay ◽  
Whole Cell ◽  
Automated Patch Clamp ◽  
Induced Currents

Abstract Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions. Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress. The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34. Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. Disclosures No relevant conflicts of interest to declare.

Cardiac Ion Channel Safety Profiling on the IonWorks Quattro Automated Patch Clamp System

2010 ◽  
Vol 8 (6) ◽  
pp. 766-780 ◽  
Author(s):  
Xueying Cao ◽  
Yan Tony Lee ◽  
Mats Holmqvist ◽  
Yingxin Lin ◽  
Yucheng Ni ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Automated Patch Clamp

scholarly journals Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chai-Ann Ng ◽  
Jessica Farr ◽  
Paul Young ◽  
Monique J Windley ◽  
Matthew D Perry ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Lines ◽  
High Throughput ◽  
Rapid Assessment ◽  
Cost Effective ◽  
Missense Variant ◽  
Loss Of Function ◽  
Channel Expression ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology

Abstract 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 bi-cistronic vector to co-express Wild-type (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 h 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.

Biophysical and pharmacological characterization of the CiPA ion channel panel and iPSC cardiomyocytes using automated patch-clamp

2016 ◽  
Vol 81 ◽  
pp. 347
Author(s):  
Olaf Scheel ◽  
Stefanie Frech ◽  
Bogdan Amuzescu ◽  
William Crumb ◽  
Mike Roberts ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Pharmacological Characterization ◽  
Automated Patch Clamp

scholarly journals Development and Validation of ASIC1a Ligand-Gated Ion Channel Drug Discovery Assays on Automated Patch Clamp Platforms

2021 ◽  
Vol 120 (3) ◽  
pp. 338a
Author(s):  
Marc Rogers ◽  
Sam Manyweathers ◽  
Tom Götze ◽  
Nadine Becker ◽  
Ilka Rinke ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Patch Clamp ◽  
Ion Channel ◽  
Automated Patch Clamp ◽  
Development And Validation
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