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Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca2+-Activated K+ Channels
Department of Assay Development and Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom
Department of Gene Expression and Protein Biochemistry, Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom
Department of Assay Development and Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom 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 (IonWorksHT) 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
This version was published on February
1, 2007 Journal of Biomolecular Screening, Vol. 12, No. 1,
50-60 (2007) This article has been cited by other articles:
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