Endothelial LRRC8C Associates With LRRC8A and LRRC8B to Regulate Vascular Reactivity and Blood Pressure

Endothelial mechanosensing is essential for controlling vascular tone. LRRC8A (leucine-rich repeat-containing protein 8A) was previously identified as a core subunit of the mechanoresponsive LRRC8 complex, functionally encoding the endothelial volume regulatory anion channel and regulating vascular function. This study aims to identify the molecular identity of the endothelial LRRC8 complex and its function in vascular reactivity and blood pressure control.

We generated germline epitope-tagged Lrrc8a-3xFlag knock-in mice and endothelium-specific Lrrc8a-3xFlag overexpression mice to permit LRRC8A and LRRC8C immunoprecipitation and define LRRC8 subunit interactions. We combined in vivo and in vitro loss-of-function models, electrophysiology, immunoblotting, and pressure myography of third-order mesenteric arteries to examine the contributions of individual LRRC8A/B/C subunits to vascular function and underlying signaling pathways. The contributions of LRRC8C to blood pressure control in vivo were further assessed using the angiotensin-induced hypertension model in Lrrc8c knockout mice.

Although all LRRC8A-E subunits are expressed in endothelium, co-immunoprecipitation revealed enrichment of LRRC8A/B/C, suggesting the existence of an endothelial LRRC8A/B/C heteromer. Lrrc8a/b/c depletion studies showed codependent expression of LRRC8A/B/C, but not LRRC8D. Only LRRC8A and LRRC8C deficiency impaired AKT and endothelial NO synthase phosphorylation, increased myogenic tone (2.2- and 1.9-fold increase, respectively), and reduced endothelial NO synthase-dependent vasodilation (45% and 61% reduction, respectively). Global Lrrc8c knockout mice phenocopied Lrrc8a knockouts and exhibited exacerbated angiotensin-induced hypertension, as evidenced by 15% increase in mean arterial pressure.

LRRC8A/B/C form the endothelial LRRC8 heteromeric complex. LRRC8C is nonredundant in supporting endothelial AKT-endothelial NO synthase signaling, vascular relaxation, and resistance to hypertension.