Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca²⁺-Activated K⁺ Channels

¹ Department of Assay Development, Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom.
² Department of Gene Expression and Protein Biochemistry, Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom.

^* To whom correspondence should be addressed. E-mail: djt26940{at}gsk.com.

	Abstract

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca²⁺-activated K⁺ channels. In CHO-hIK--expressing cells, under asymmetric K⁺ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC₅₀ values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC₅₀ values of 0.31 µM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca²⁺-activated K⁺ channels and provides significant advantages over single-cell electrophysiology (IonWorks^HT) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non--voltage-gated ion channels.

Key Words: planar array electrophysiology, population patch clamp, Ca²⁺-activated K⁺ channel, apamin, TRAM-34

First published on November 7, 2006, doi:10.1177/1087057106294920

Journal of Biomolecular Screening 2007;12:50.

A more recent version of this article appeared on February 1, 2007


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