Endothelin-1 Stimulates Vasoconstriction Through Rab11A Serine 177 Phosphorylation

Abstract

Rationale: Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BKα and auxiliary β1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BKα is primarily plasma membrane localized, whereas the cellular location of β1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. Objective: Test the hypothesis that ET-1 inhibits BK channels by altering BKα and β1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. Methods and Results: ET-1, through activation of PKC (protein kinase C), reduced surface β1 abundance and the proximity of β1 to surface BKα in myocytes. In contrast, ET-1 did not alter surface BKα, total β1, or total BKα proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1–induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface β1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of β1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. Conclusions: ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of β1 subunits. The decrease in surface β1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.