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Endothelial cells (ECs) are electrically coupled to arterial smooth muscle cells in the vascular wall and can modulate their contractility through direct control of membrane potential and via the production of several vasoactive substances. ECs express several cation channels which are known to regulate arterial contractility. In contrast, physiological functions of anion channels in ECs are unclear. ECs express TMEM16A, a Ca2+-activated Cl-channel. To examine physiological functions of TMEM16A channels in ECs, we generated an inducible, EC-specific TMEM16A knockout (TMEM16A ecKO) mouse. An increase in intracellular Ca2+ concentration ([Ca2+]i) or the application of acetylcholine (ACh) activated Cl‑ currents in fresh-isolated mesenteric artery ECs of control ( TMEM16Afl/fl) mice that were inhibited by tannic acid or benzbromarone, which are TMEM16A channel blockers. In contrast, an increase in [Ca2+]i or ACh did not activate Cl‑ currents in TMEM16A ecKO ECs. HC067047, a TRPV4 channel blocker, inhibited TMEM16A current activation by ACh in TMEM16Afl/fl ECs. GSK101, a TRPV4 channel activator, stimulated TMEM16A currents in TMEM16Afl/fl ECs, but not in TMEM16A ecKO ECs. Super resolution single-molecule localization microscopy demonstrated that surface clusters of TMEM16A and TRPV4 channels locate in nanoscale spatial proximity in the plasma membrane of ECs. ACh produced membrane hyperpolarization in pressurized TMEM16Afl/fl arteries that was attenuated in TMEM16A ecKO arteries. Vasodilation to ACh was smaller in pressurized TMEM16A ecKO arteries than in TMEM16Afl/fl arteries. TMEM16A knockout in ECs increased blood pressure in mice. These data indicate that ACh activates TRPV4-coupled TMEM16A channels in ECs to induce membrane hyperpolarization, vasodilation and a reduction in blood pressure. National Institutes of Health This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.