title: Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.
abstract: The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos 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 responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas 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 Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) 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.
authors.affiliation: 1 Department of Pharmacology, University of California , Davis, California.
authors.forename: David Paul
authors.initials: DP
authors.lastname: Jenkins
authors.forename: Weifeng
authors.initials: W
authors.lastname: Yu
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Brown
authors.forename: Lars Damgaard
authors.initials: LD
authors.lastname: Løjkner
authors.forename: Heike
authors.initials: H
authors.lastname: Wulff
mesh_terms: D001681:Biological Assay / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000502:physiology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D012371:Robotics / Q000379:methods
chemical_list: D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D026902:Potassium Channel Blockers