Manipulation of smooth muscle BKCa using subunit directed siRNA

Abstract

Cremaster and cerebral vascular smooth muscle cells (VSMC) exhibit heterogeneity in large conductance Ca2+‐activated K+ channels (BKCa) partly due to differences in β1:α subunit ratio. To gain insight into BKCa methods were developed for subunit‐specific knockdown of the channel. Using transient transfection approaches and small interfering RNAs (siRNA) either the α or β1 subunit was targeted in isolated arterioles. Control studies used fluorescently labeled siRNA or unrelated siRNA. After 2–3 days culture fluorescence images and whole cell K+ currents were examined in dispersed VSMC. From functional data α‐subunit expression was reduced by ~60% in both vessels. Thus, at +70 mV, IBTX‐sensitive K+ current density was significantly reduced after α‐subunit siRNA compared to control. Similarly, STOC frequency (at +20 mV) decreased following siRNA treatment while BKCa opening by NS1619 or estrogen (E2) was decreased. Cells treated with β1‐subunit siRNA showed impaired responses to E2 with a greater effect in cerebral VSMCs compared to those of cremaster. Thus transient transfection and siRNA can be used to effectively decrease endogenous BKCa activity in intact small arteries. Further, cerebral VSMCs treated with β1‐subunit siRNA exhibit a functional phenotype similar to untreated cremaster VSMCs, supporting the idea that differences in β1:α subunit ratio underlie observed heterogeneity in BKCa activity.