Endothelin-1 Stimulates Vasoconstriction Through Rab11A Serine 177 Phosphorylation
Rationale: Large-conductance calcium (Ca2+)-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 multi-subunit 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 protein kinase C (PKC), 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, nitric oxide-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 Ca2+-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.
- Received April 4, 2017.
- Revision received July 6, 2017.
- Accepted July 7, 2017.