The suitability of high throughput automated patch clamp for physiological applications

• Published in: The Journal of physiology Vol. 600; no. 2; pp. 277 - 297
• Main Authors: Obergrussberger, Alison, Rinke‐Weiß, Ilka, Goetze, Tom A., Rapedius, Markus, Brinkwirth, Nina, Becker, Nadine, Rotordam, Maria Giustina, Hutchison, Laura, Madau, Paola, Pau, Davide, Dalrymple, David, Braun, Nina, Friis, Søren, Pless, Stephan A., Fertig, Niels
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.01.2022
• Subjects: Action potential; Allosteric properties; automated patch clamp; Automation; Calcium (intracellular); Cardiomyocytes; cell lines; Drug Discovery; Flow cytometry; Genetic diversity; Glutamic acid receptors (ionotropic); High-Throughput Screening Assays; Intracellular; Ion Channels; Ion channels (cyclic nucleotide-gated); ligand‐gated ion channels; Myocytes, Cardiac; N-Methyl-D-aspartic acid receptors; Patch-Clamp Techniques; Sodium channels (voltage-gated); Stem cells; voltage‐gated ion channels; automated patch clamp; stem cells; voltage-gated ion channels; ligand-gated ion channels; cell lines; ion channels
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/JP282107

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 gigaohm 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 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. 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 fluorescence‐activated cell sorting (FACS) prior to APC experiments. Gigaohm 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, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. Key points High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384‐well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell‐derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode. figure legend Different cell types including stable cell lines, transfected cells, stem cells and primary cells are used on automated patch clamp to generate high throughput pharmacology data, for mutant investigations and clone selection, and functional analysis of ion channels. Created with BioRender.com.