TY - JOUR

T1 - The suitability of high throughput automated patch clamp for physiological applications

AU - Obergrussberger, Alison

AU - Rinke-Weiß, Ilka

AU - Goetze, Tom A.

AU - Rapedius, Markus

AU - Brinkwirth, Nina

AU - Becker, Nadine

AU - Rotordam, Maria Giustina

AU - Hutchison, Laura

AU - Madau, Paola

AU - Pau, Davide

AU - Dalrymple, David

AU - Braun, Nina

AU - Friis, Søren

AU - Pless, Stephan A.

AU - Fertig, Niels

N1 - Funding Information: We acknowledge the Lundbeck Foundation (R139‐2012‐12390 to S.A.P. and R218‐2016‐1490 to N.B.) and the Boehringer Ingelheim Fond (to N.B.) for financial support. Publisher Copyright: © 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

PY - 2022

Y1 - 2022

N2 - Abstract: 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.

AB - Abstract: 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.

KW - automated patch clamp

KW - cell lines

KW - ion channels

KW - ligand-gated ion channels

KW - stem cells

KW - voltage-gated ion channels

U2 - 10.1113/JP282107

DO - 10.1113/JP282107

M3 - Journal article

C2 - 34555195

AN - SCOPUS:85116598919

VL - 600

SP - 277

EP - 297

JO - The Journal of Physiology

JF - The Journal of Physiology

SN - 0022-3751

IS - 2

ER -