Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca2+-Activated K+ Channels
| Autor: | Emma C. Hollands, Derek J. Trezise, Victoria H John, David L. Downie, Mao Xiang Chen, Tim Dale, Leanne Partington, Helen J. Meadows |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Patch-Clamp Techniques
Patch clamp electrophysiology Population Analytical chemistry CHO Cells Apamin Biochemistry Analytical Chemistry law.invention Potassium Channels Calcium-Activated chemistry.chemical_compound Cricetulus law Cricetinae Membrane Transport Modulators Animals Humans Patch clamp education K channels education.field_of_study Dose-Response Relationship Drug Activator (genetics) Chemistry Reproducibility of Results Electrophysiology Biophysics Recombinant DNA Molecular Medicine Biotechnology |
| Zdroj: | SLAS Discovery. 12:50-60 |
| ISSN: | 2472-5552 |
| DOI: | 10.1177/1087057106294920 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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