Endothelial LRRC8C associates with LRRC8A and LRRC8B to regulate vascular reactivity and blood pressure

Vascular tone is impacted by the endothelium's ability to detect mechanical and chemical stimulation. <i>L</i> eucine- <i>R</i> ich <i>R</i> epeat- <i>C</i> ontaining protein <i>8</i> A, (LRRC8A), was previously identified as a required component of the mechanoresponsive endothelial LRRC8 complex regulating AKT-endothelial nitric oxide synthase (eNOS) signaling and vascular function. While LRRC8A is broadly expressed, LRRC8B, C, D and E have tissue-restricted expression. Here, we identified 2 single nucleotide polymorphisms (SNPs) in <i>LRRC8C</i> highly associated with elevated diastolic and systolic blood pressure in human genetic studies, implicating LRRC8C as a regulator of vascular function. While LRRC8A/B/C/D/E are expressed in endothelium, co-immunoprecipitation experiments from lung endothelium using <i>Lrrc8a</i> -3xFlag knock-in mice, <i>Lrrc8c-</i> HA knock-in mice and endothelium-specific <i>Lrrc8a</i> -3xFlag overexpression mice demonstrate the endothelial LRRC8 complex to be composed largely of LRRC8A/B/C heteromers. <i>Lrrc8a/b/c</i> knock-out studies in mice and knock-down studies in human umbilical vein endothelial cells show co-dependent expression of LRRC8A/B/C proteins, but not LRRC8D. Functionally, LRRC8A and LRRC8C depletion reduces endothelial volume regulatory anion channel (VRAC) currents, inhibits AKT-eNOS signaling, increases myogenic tone, impairs eNOS dependent vasodilation, and exacerbates angiotensin-induced hypertension. These data identify LRRC8A, LRRC8B and LRRC8C as components of the endothelial LRRC8 complex and reveal LRRC8C as having a non-redundant role in regulating endothelial AKT-eNOS, vascular relaxation and susceptibility to hypertension.