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.

原理：大传导钙激活钾通道（BK）由动脉平滑肌细胞（肌细胞）中的成孔BKα和辅助β1亚基组成。血管收缩剂，包括内皮素-1（ET-1），抑制肌细胞BK通道，导致收缩，但涉及的机制尚不清楚。最近的证据表明，BKα主要位于质膜上，而β1的细胞位置可以被Rab11A阳性回收内体快速改变。血管收缩剂是否调节表面BK通道的多亚基组成以刺激收缩尚不清楚。

目的：检验ET-1通过改变心肌细胞中BKα和β1表面转运来抑制BK通道的假说，确定涉及的机制，并确定小脑动脉心肌细胞的功能意义。

方法和结果：ET-1，通过激活PKC（蛋白激酶C），减少了心肌细胞表面β1的丰度以及β1与表面BKα的接近度。相反，ET-1不会改变表面BKα，总β1或总BKα蛋白。ET-1刺激Rab11A磷酸化，从而降低Rab11A活性。Rab11A丝氨酸177被确定为高概率PKC磷酸化位点。无法磷酸化的Rab11A构建体（Rab11A S177A）的表达阻断了ET-1诱导的Rab11A磷酸化，Rab11A活性降低和表面β1蛋白降低。ET-1通过需要Rab11A S177的PKC依赖性机制抑制了心肌细胞中的单个BK通道和瞬时BK电流，并刺激了血管收缩。相反，NO诱导的Rab11A激活，β1亚基的表面转运，BK通道和瞬时BK电流激活，

结论： ET-1刺激丝氨酸177处PKC介导的Rab11A磷酸化，从而抑制了依赖Rab11A和Rab11A依赖性的β1亚基的表面转运。表面β1亚基的减少导致BK通道钙敏感性降低，抑制瞬时BK电流和血管收缩。我们描述了一种独特的机制，血管收缩剂可通过该机制抑制BK通道，并将Rab11A丝氨酸177识别为动脉收缩性的调节剂。