Spatiotemporal Control of Vascular Ca_V1.2 by α1_C S1928 Phosphorylation.

Abstract

L-type Ca_V1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the Ca_V1.2 pore-forming subunit α1_C at S1928 mediates vascular Ca_V1.2 cooperativity during diabetic hyperglycemia. A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine Ca_V1.2 cooperativity, α1_C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. Ca_V1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1_C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y₁₁ inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. Ca_V1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca²⁺ influx and contractility and <i>in vivo</i> reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. These results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1_C into "superclusters" upon hyperglycemia and diabetes. This triggers Ca_V1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct Ca_V1.2 and arterial function during diabetic hyperglycemia.