Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.
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.
John
Victoria H
VH
Department of Assay Development, Discovery Research Biology, GlaxoSmithKline Research & Development, Gunnels Wood Road, Stevenage, Hertfordshire, SG7 5NJ, United Kingdom.
Dale
Tim J
TJ
Hollands
Emma C
EC
Chen
Mao Xiang
MX
Partington
Leanne
L
Downie
David L
DL
Meadows
Helen J
HJ
Trezise
Derek J
DJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000379:methods*; D006801:Humans; D049990:Membrane Transport Modulators / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods*; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results
D049990:Membrane Transport Modulators; D024681:Potassium Channels, Calcium-Activated