Membrane Potential Controls Surface Expression of BK Channel Auxiliary β1 Subunits in Arterial Smooth Muscle Cells

Abstract

Intravascular pressure stimulates arterial depolarization, leading to smooth muscle cell (myocyte) voltage‐dependent calcium (Ca2+) channel activation and vasoconstriction. Membrane depolarization also activates BK channels, which oppose pressure‐induced depolarization and constriction, but mechanisms involved are unclear. We have previously shown that nitric oxide stimulates rapid surface expression of β1 subunits in arterial myocytes. These β1 subunits activate surface‐resident BK channels, leading to vasodilation. Here, we tested the novel hypothesis that membrane potential controls β1 trafficking to regulate BK channel activity. Using techniques, including surface biotinylation, immuno‐FRET microscopy, western blotting and gene knockdown, we show that membrane depolarization (30–60 mM K+ PSS) rapidly (<10 min) increased the surface abundance of BK channel auxiliary β1 subunits in cerebral artery myocytes. Depolarization‐induced β1 trafficking was inhibited by: 1) brefeldin A, a trafficking inhibitor, 2) knockdown of rab11A, a small GTPase associated with recycling endosomes, 3) rab11A dominant negative mutant overexpression, 4) nimodipine, a CaV1.2 channel blocker, 5) HA‐1100, a rho kinase (ROCK) inhibitor, but not by KN93, a Ca2+/calmodulin‐dependent protein kinase inhibitor, or LY294002, a specific PI3 kinase inhibitor, 6) ROCK2, but not ROCK1, knockdown. HA‐1100 also reduced GTP‐bound active rab11 in arteries. These data indicate that membrane depolarization stimulates surface trafficking of β1 through a mechanism that involves voltage‐dependent Ca2+ channel and ROCK2 activation in arterial myocytes.Support or Funding InformationRO1 HL67661, HL110347, HL094378 to JHJ and American Heart Assoc.‐SDG to MDL