Endothelial CYB5R3 couples store-operated calcium entry to TRPV2 activation and vascular fitness

Abstract

NADH-cytochrome b5 reductase 3 (CYB5R3) is a flavoprotein that governs nitric oxide (NO) signaling and supports NADPH oxidase 4-derived hydrogen peroxide production via coenzyme Q reduction in endothelium. While CYB5R3 expression is decreased during aging, the downstream consequences of CYB5R3 loss are not understood. Here, we demonstrate that depletion of CYB5R3 in primary human aortic endothelial cells activates a Ca2+ influx network characterized by the upregulation of calcium release-activated calcium (CRAC) channel subunits ORAI2 and ORAI3, as well as the non-selective cation channel transient receptor potential vanilloid 2 (TRPV2). When endoplasmic-reticulum Ca2+ stores were depleted, CYB5R3-deficient cells had increased Ca2+ entry through the plasma membrane, part of which was insensitive to classical store-operated Ca2+ entry (SOCE) blockers and was mediated by TRPV2, as demonstrated by genetic knockdown and pharmacologic inhibition. Mechanistically, loss of CYB5R3 increased Ca2+-dependent NO production through elevated CRAC channel activity, which oxidatively inhibited the protein tyrosine phosphatase non-receptor type 1 (PTPN1). This prevented TRPV2 dephosphorylation, thereby maintaining Janus kinase 1 (JAK1)-dependent channel activation downstream of SOCE. It also enhanced the responsiveness of TRPV2 to physiological heat stimuli. Thus, CYB5R3 normally acts as a brake, limiting NO-dependent PTPN1 oxidation and restraining TRPV2 activity. In vivo, endothelial-specific Cyb5r3 deletion enhanced acetylcholine-induced vasorelaxation and improved exercise capacity, demonstrating a physiological function for this pathway in vascular adaptation. Together, these findings identify a CYB5R3-NO-SOCE- PTPN1-TRPV2 signaling axis that couples endothelial redox balance to Ca2+ dynamics and vascular function. SIGNIFICANCEEndothelial cells rely on receptor-regulated Ca2+ signals to produce vasodilators and control vascular function; however, the molecular mechanisms coordinating these pathways are incompletely understood. We identify CYB5R3 as a key redox switch that couples store-operated Ca2+ entry to the non-selective cation channel TRPV2. Loss of CYB5R3 enhances TRPV2 activity downstream of SOCE through NO-dependent oxidative inhibition of the phosphatase PTPN1, sustaining Janus kinase-mediated TRPV2 channel activation. This novel mechanism expands the physiological scope of CYB5R3 by redefining how redox enzymes intersect with Ca2+ signaling, linking endothelial CYB5R3 to vascular relaxation and exercise capacity in vivo. This positions CYB5R3 as a central regulator of vascular function with broad implications for cardiovascular health and disease.