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.
Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)–activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK β1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of β1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional β1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of β1 subunits to the surface of arterial myocytes.
Hypertension is a risk factor for cerebrovascular diseases, including stroke and dementia. During hypertension, arteries become constricted and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by smooth muscle cell (myocyte) large-conductance calcium (Ca)-activated potassium (BK) channels is altered during hypertension, although mechanisms involved are unclear. We tested the hypothesis that dysfunctional trafficking of pore-forming BK channel (BKα) and auxiliary β1 subunits contributes to changes in cerebral artery contractility of stroke-prone spontaneously hypertensive rats (SP-SHRs). Our data indicate that the amounts of total and surface BKα and β1 proteins are similar in unstimulated arteries of age-matched SP-SHRs and normotensive Wistar-Kyoto rats. In contrast, stimulated surface-trafficking of β1 subunits by NO or membrane depolarization is inhibited in SP-SHR myocytes. PKCα (protein kinase C α) and PKCβII total protein and activity were both higher in SP-SHR than in Wistar-Kyoto rat arteries. NO or depolarization robustly activated Rab11, a small trafficking GTPase, in Wistar-Kyoto rat arteries but weakly activated Rab11 in SP-SHRs. Bisindolylmaleimide, a PKC inhibitor, and overexpression of a PKC phosphorylation-deficient Rab11A mutant (Rab11A S177A) restored stimulated β1 subunit surface-trafficking in SP-SHR myocytes. BK channel activation by NO was inhibited in SP-SHR myocytes and restored by Rab11A S177A expression. Vasodilation to NO and lithocholate, a BKα/β1 channel activator, was inhibited in pressurized SP-SHR arteries and reestablished by bisindolylmaleimide. In summary, data indicate that spontaneously active PKC inhibits Rab11A-mediated β1 subunit trafficking in arterial myocytes of SP-SHRs, leading to dysfunctional NO-induced BK channel activation and vasodilation.
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