The suitability of high throughput automated patch clamp for physiological applications.

Abstract

KEY POINTS\nHigh throughput automated patch clamp can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus FLIPRTM in a high throughput compound screen against NMDA receptors. \xa0Genetic 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.\n\n\nABSTRACT\nAlthough 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 GΩ 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 1,920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPRTM . Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 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 FACS prior to APC experiments. GΩ 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, this highlights the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. Abstract 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. This article is protected by copyright. All rights reserved.