Screening Assay Protocols Targeting the Nav1.7 Channel Using Qube High‐Throughput Automated Patch‐Clamp System

2020 ◽  
Vol 89 (1) ◽  
Author(s):  
Binbin Qian ◽  
Sung Hoon Park ◽  
Weifeng Yu
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
Screening Assay ◽  
Automated Patch Clamp

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.

scholarly journals Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

2016 ◽  
Vol 37 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Jin-feng Yue ◽  
Guan-hua Qiao ◽  
Ni Liu ◽  
Fa-jun Nan ◽  
Zhao-bing Gao
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Techniques ◽  
Automated Patch Clamp

scholarly journals High Throughput Pharmacology of Cardiac L-Type Ca2+ Channels: Stable Recordings of CaV1.2 on a Highly Parallel Automated Patch Clamp System

2016 ◽  
Vol 110 (3) ◽  
pp. 443a-444a ◽  
Author(s):  
Markus Rapedius ◽  
Andrea Bruggemann ◽  
Tom Goetze ◽  
Claudia Haarmann ◽  
Ilka Rinke ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
Automated Patch Clamp ◽  
Ca2 Channels

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.

scholarly journals Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0243645
Author(s):  
Md. Mahadhi Hasan ◽  
Lotten Ragnarsson ◽  
Fernanda C. Cardoso ◽  
Richard J. Lewis
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
Mammalian Cells ◽  
Imaging Plate ◽  
Voltage Gated ◽  
Cellular Assays ◽  
Pain Pathways ◽  
Automated Patch Clamp ◽  
Voltage Gated Ion Channels ◽  
And Function

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

scholarly journals Rapid characterisation of hERG channel kinetics I: using an automated high-throughput system

2019 ◽  
Author(s):  
Chon Lok Lei ◽  
Michael Clerx ◽  
David J. Gavaghan ◽  
Liudmila Polonchuk ◽  
Gary R. Mirams ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Voltage Clamp ◽  
High Throughput ◽  
Rapid Development ◽  
Herg Channel ◽  
Model Parameters ◽  
Channel Kinetics ◽  
Automated Patch Clamp ◽  
High Information

ABSTRACTPredicting 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 vitrohERG-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 SignificanceWe 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.

scholarly journals The suitability of high throughput automated patch clamp for physiological applications

2021 ◽  
Author(s):  
Alison Obergrussberger ◽  
Ilka Rinke‐Weiß ◽  
Tom A Goetze ◽  
Markus Rapedius ◽  
Nina Brinkwirth ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
Automated Patch Clamp

scholarly journals Cover Picture: Identification of Selective Inhibitors of the Potassium Channel Kv1.1-1.2(3)by High-Throughput Virtual Screening and Automated Patch Clamp (ChemMedChem 10/2012)

ChemMedChem ◽  
2012 ◽  
Vol 7 (10) ◽  
pp. 1693-1693
Author(s):  
Sören J. Wacker ◽  
Wiktor Jurkowski ◽  
Katie J. Simmons ◽  
Colin W. G. Fishwick ◽  
A. Peter Johnson ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Patch Clamp ◽  
Potassium Channel ◽  
High Throughput ◽  
Selective Inhibitors ◽  
Automated Patch Clamp ◽  
Picture Identification ◽  
High Throughput Virtual Screening
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