Project Summary Endothelial cells line the wall of all blood vessels and regulate a wide variety of functions, including contractility and systemic blood pressure. Endothelial cell dysfunction is a hallmark of several cardiovascular diseases, but pathological mechanisms involved are unclear. Endothelial cells express small-conductance calcium-activated potassium (SK3) channels, which regulate contractility. Currents (I) generated by SK3 channels in endothelial cells is the product of the number of channels (N), their open probability (PO) and amplitude (i), such that I=N.PO.i. Previous studies have focused on identifying mechanisms that regulate the activity of surface SK channels in endothelial cells. In contrast, mechanisms that control the number (N) of surface SK3 channels in endothelial cells are poorly understood. Importantly, it is unclear whether physiological stimuli regulate the abundance of surface SK3 channels to alter arterial contractility. Similarly uncertain is if pathological alterations in the control of surface SK3 channel abundance occurs during hypertension, leading to attenuated vasodilation. Using a wide variety of approaches, we provide evidence that vasodilator stimuli activate trafficking mechanisms that rapidly increase surface SK3 channel abundance in endothelial cells to induce vasodilation. Preliminary data also suggest that SK3 channel trafficking is dysfunctional during hypertension, which attenuates this vasodilatory signaling mechanism. In this proposal, we will investigate three specific aims. Aim 1 will investigate signaling mechanisms by which physiological stimuli alter the surface abundance of SK3 channels and examine their functional significance. Aim 2 will examine trafficking mechanisms that control surface SK3 channels in endothelial cells to modify arterial contractility. Aim 3 will study the hypothesis that hypertension is associated with pathological alterations in SK3 channel trafficking in endothelial cells that inhibit vasodilation by these proteins. Methods used will include biotinylation, Western blotting, FRET, RNAi, co-IP, immunofluorescence, super-resolution microscopy, patch-clamp electrophysiology, membrane potential recording, intracellular Ca2+ imaging, arterial myography, blood pressure telemetry and transgenic mice. This project will provide significant novel information concerning vasoregulation by SK3 channel trafficking in endothelial cells.