Vasodilators stimulate SK3 channel anterograde trafficking in endothelial cells to elicit vasodilation

Endothelial cells (ECs) line the lumen of blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intraluminal flow, activate small conductance calcium-activated potassium (SK3) channels in ECs, leading to hyperpolarization and vasodilation. Whether these stimuli modulate SK3 surface abundance in ECs to elicit vasodilation is unclear. Here, we investigated physiological regulation of SK3 channel surface trafficking in mesenteric artery (MA) ECs. Biotinylation experiments demonstrated that ACh or intravascular flow rapidly (2 min) increased surface SK3 protein ~ 2.0- and 2.1-fold, respectively in MAs, with similar results obtained in primary-cultured ECs. Brefeldin A, an anterograde trafficking inhibitor, removal of extracellular Ca2+ , GSK2193874, a selective TRPV4 channel blocker or TRPV4 knockdown using siRNA, each inhibited the ACh-induced increase in surface SK3 channels in MAs. Super resolution microscopy (SMLM) experiments demonstrated that ACh increased the size of surface SK3 clusters in ECs. ACh activated whole-cell SK currents and this effect was inhibited by brefeldin A. Brefeldin A also reduced vasodilation induced by ACh and intravascular flow to between ~48 and 57 % of those in control. Brefeldin A blocked the SK channel component of ACh- and flow-mediated vasodilations. In summary, our data indicate that ACh and flow stimulate anterograde trafficking of SK3 channels in ECs via a TRPV4-dependent Ca2+ signaling mechanism to elicit vasodilation.