Development of a Qpatch Automated Electrophysiology Assay for Identifying Kca3.1 Inhibitors and Activators

Abstract

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 (also known as KCNN4, IK1 or the Gardos channel) plays an important role in the activation of T- and B-cells, mast cells, macrophages and microglia by regulating membrane potential, cellular volume and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization (EDH) responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorder, while KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca2+ sensitivity of KCa3.1 was studied using varying intracellular Ca2+ concentrations. A free Ca2+ concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca2+ concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.Supported by U54NS079202, R21NS072585 and T32-GM008799 from NIH.