Control of Arterial Ca V 1.2 Clustering and Coupled Gating by a Single Amino Acid

Abstract

The L-type Ca2+ channel CaV1.2 plays key roles in cell excitability, muscle contraction and gene expression. These channels have been shown to gate in unison (e.g. coupled gating) in several cell types. CaV1.2 coupling amplifies Ca2+ influx, which can regulate cell function. In arterial myocytes, CaV1.2 coupling is increased upon elevations in extracellular glucose (HG) and in cells from diabetic patients and animal models of diabetes. Yet, the mechanisms for induction of this gating modality in CaV1.2 channels are poorly understood. Here, we tested the hypothesis that phosphorylation of CaV1.2 at S1928 is critical for induction of CaV1.2 clustering, which contributes to CaV1.2 coupling, amplification of Ca2+ influx and arterial myocyte contraction in response to HG and diabetes. Using a multiscale approach, we found that HG induced an increase in CaV1.2-mediated activity, open probability, and coupling frequency and strength in WT arterial myocytes, but not cells from a mouse in which S1928 was mutated to alanine to prevent the phosphorylation of this site (e.g. S1928A). Wild type-like CaV1.2 response to HG was observed in arterial myocytes from a mouse in which S1700 (another key CaV1.2 phosphorylation site) was prevented (e.g. S1700A), thus underscoring the importance S1928 phosphorylation on CaV1.2 function. Super-resolution nanoscopy and proximity ligation assay (PLA) revealed an increase in CaV1.2 clustering at the surface membrane of WT, but not S1928A arterial myocytes after acute HG incubation. Similar HG-induced CaV1.2 biophysical and structural alterations were recapitulated in arterial myocytes from WT diabetic mice (streptozotocin model) and human with diabetes, and these changes were completely absent in cells from diabetic S1928A mice. These results suggest a key role for S1928 phosphorylation in modulating CaV1.2 spatial distribution and gating mode upon HG and diabetes. We propose that our work may lay the foundation for novel therapeutic strategies with single amino acid accuracy to correct channel function and vascular reactivity during pathological conditions.