title: The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7.
abstract: The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca(2+) permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms-the IonWorks(®) Quattro™ and QPatch(®) systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.
authors.affiliation: Novartis Institutes for BioMedical Research , Horsham Research Centre, West Sussex, United Kingdom .
authors.forename: Mark
authors.initials: M
authors.lastname: McPate
authors.forename: Gurdip
authors.initials: G
authors.lastname: Bhalay
authors.forename: Martin
authors.initials: M
authors.lastname: Beckett
authors.forename: Sian
authors.initials: S
authors.lastname: Fairbrother
authors.forename: Martin
authors.initials: M
authors.lastname: Gosling
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Groot-Kormelink
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lane
authors.forename: Toby
authors.initials: T
authors.lastname: Kent
authors.forename: Michiel T
authors.initials: MT
authors.lastname: Van Diepen
authors.forename: Pamela
authors.initials: P
authors.lastname: Tranter
authors.forename: J Martin
authors.initials: JM
authors.lastname: Verkuyl
mesh_terms: D000818:Animals; D001331:Automation; D016466:CHO Cells; D002478:Cells, Cultured; D003412:Cricetulus; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D018408:Patch-Clamp Techniques / Q000379:methods*; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C498501:TRPC7 protein, human