High-Throughput Screening of NaV1.7 Modulators Using a Giga-Seal Automated Patch Clamp Instrument

2016 ◽  
Vol 14 (2) ◽  
pp. 93-108 ◽  
Author(s):  
Chris Chambers ◽  
Ian Witton ◽  
Cathryn Adams ◽  
Luke Marrington ◽  
Juha Kammonen
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
High Throughput Screening ◽  
Automated Patch Clamp

scholarly journals Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module

2015 ◽  
Vol 21 (6) ◽  
pp. 779-793 ◽  
Author(s):  
A. Obergrussberger ◽  
A. Bru ggemann ◽  
T. A. Goetze ◽  
M. Rapedius ◽  
C. Haarmann ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput ◽  
High Throughput Screening ◽  
Automated Patch Clamp

scholarly journals An advanced automated patch clamp protocol design to investigate drug - ion channel binding dynamics.

2021 ◽  
Author(s):  
Peter Lukacs ◽  
Krisztina Pesti ◽  
Matyas C Foldi ◽  
Katalin Zboray ◽  
Adam V Toth ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Mechanism Of Action ◽  
Time Scale ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Neuromuscular Disorders ◽  
Rapid Assessment ◽  
Drug Effects ◽  
Dissociation Kinetics ◽  
Automated Patch Clamp

Standard high throughput screening projects using automated patch-clamp instruments often fail to grasp essential details of the mechanism of action, such as binding/unbinding dynamics and modulation of gating. In this study, we aim to demonstrate that depth of analysis can be combined with acceptable throughput on such instruments. Using the microfluidics-based automated patch clamp, IonFlux Mercury, we developed a method for a rapid assessment of the mechanism of action of sodium channel inhibitors, including their state-dependent association and dissociation kinetics. The method is based on a complex voltage protocol, which is repeated at 1 Hz. Using this time resolution we could monitor the onset and offset of both channel block and modulation of gating upon drug perfusion and washout. Our results show that the onset and the offset of drug effects are complex processes, involving several steps, which may occur on different time scales. We could identify distinct sub-processes on the millisecond time scale, as well as on the second time scale. Automated analysis of the results allows collection of detailed information regarding the mechanism of action of individual compounds, which may help the assessment of therapeutic potential for hyperexcitability-related disorders, such as epilepsies, pain syndromes, neuromuscular disorders, or neurodegenerative diseases.

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.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors

2020 ◽  
Vol 25 (5) ◽  
pp. 434-446
Author(s):  
Hongkang Zhang ◽  
Bryan D. Moyer ◽  
Violeta Yu ◽  
Joseph G. McGivern ◽  
Michael Jarosh ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
High Reliability ◽  
Selective Inhibitors ◽  
New Class ◽  
Random Library ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology ◽  
Screening Platform ◽  
Subtype Selective

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

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.

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