Project Summary Endothelial cells regulate a wide variety of vascular functions, including contractility, which modulates organ blood flow and systemic blood pressure. A hallmark of virtually all cardiovascular diseases is dysfunctional endothelial cells, but pathological mechanisms involved are uncertain. Several cation channels are expressed in ECs that modulate arterial contractility, including small- (SK) and intermediate (IK)-conductance calcium (Ca2+)-activated potassium channels and transient receptor potential channels, including TRPV4. In contrast, physiological functions of anion channels in ECs are poorly understood. ECs express TMEM16A, a Ca2+- activated Cl- channel, but whether this anion channel regulates arterial contractility is unclear. Similarly uncertain is the pathological involvement of endothelial cell TMEM16A channels in the vascular dysfunction that occurs during hypertension. Using a broad range of approaches, including tamoxifen-inducible endothelial cell-specific knockout mice, we provide evidence that vasodilator stimuli activate TMEM16A channels in endothelial cells to induce vasodilation. Preliminary data also suggest that endothelial cell TMEM16A channels are dysfunctional during hypertension. In this proposal, we will investigate three specific aims. Aim 1 will investigate the signaling mechanisms by which physiological vasodilators activate TMEM16A channels in ECs to induce vasorelaxation. Aim 2 will examine the mechanisms by which the activation of TMEM16A channels in endothelial cells elicits vasodilation. Aim 3 will study the hypothesis that hypertension is associated with pathological alterations in TMEM16A channels in endothelial cells which inhibits vasodilation by these proteins. Methods used will include molecular biology and biochemistry techniques, super-resolution immunofluorescence microscopy, high-speed dual-inverted selective plane illumination microscopy (diSPIM), two-photon confocal microscopy, patch-clamp and conventional electrophysiology, arterial myography and radiotelemetry. This project will provide significant novel information regarding physiological and pathological vasoregulation by TMEM16A channels in endothelial cells.