Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels

• Published in: Journal of biomolecular screening Vol. 12; no. 1; p. 50
• Main Authors: John, Victoria H, Dale, Tim J, Hollands, Emma C, Chen, Mao Xiang, Partington, Leanne, Downie, David L, Meadows, Helen J, Trezise, Derek J
• Format: Journal Article
• Language: English
• Published: United States 01-02-2007
• Subjects: Animals; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Electrophysiology - methods; Humans; Membrane Transport Modulators - pharmacology; Patch-Clamp Techniques - methods; Potassium Channels, Calcium-Activated - antagonists & inhibitors; Potassium Channels, Calcium-Activated - metabolism; Reproducibility of Results
• ISSN: 1087-0571
• DOI: 10.1177/1087057106294920

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 microM 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.