WNK kinase is a vasoactive chloride sensor in endothelial cells

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl^-) is the most abundant anion in ECs and the Cl^- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl^- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl^- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl^- concentration ([Cl^-]_i) due to the activation of TMEM16A, a Cl^- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl^- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca²⁺ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca²⁺ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl^-]_i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl^- signaling and WNK kinase activity in ECs to control arterial contractility.