Spatiotemporal Control of Vascular Ca _V 1.2 by α1 _C S1928 Phosphorylation

Abstract

BACKGROUND: L-type Ca V 1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the Ca V 1.2 pore-forming subunit α1 C at S1928 mediates vascular Ca V 1.2 cooperativity during diabetic hyperglycemia. METHODS: A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, pressure myography, and Laser Speckle imaging was implemented to examine Ca V 1.2 cooperativity, α1 C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. RESULTS: Ca V 1.2 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 P2Y 11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. Ca V 1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca 2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. CONCLUSIONS: These results suggest that PKA-dependent pS1928 promotes the spatial reorganization of vascular α1 C into “superclusters” upon hyperglycemia and diabetes. This triggers Ca V 1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct Ca V 1.2 and arterial function during diabetic hyperglycemia.

Diversity of cells and signals in the cardiovascular system

“…A further regulatory influence over ion fluxes across the plasma membrane is the clustering state of the channels. The idea that the nanoscale arrangement of ion channels in the plasma membrane can impact their function is an emerging concept in several fields, including cardiac and vascular smooth muscle physiology (Del Villar et al., 2021; Dixon et al., 2022; Martin‐Aragon et al., 2022). At the Symposium, the concept was discussed that the clustering state of ion channels affects their activity and also extends to SR‐localized RyR2, where heterogeneity of cluster sizes has been found to support arrhythmogenic Ca 2+ ‐wave propagation (Galice et al., 2018; Xie et al., 2019).…”

Peering into the Molecular Machinery for Regulation of Cav1.2 Channel Clusters

“…More recently, our understanding of ion channel biology has continued to develop with increasing recognition and characterization of “channel clustering,” or the observation that individual ion channels can form organized colocalization with one another, leading to events such as cooperative gating and/or biophysical coupling, current augmentation, and physiologic function. In this issue of Circulation Research , Baudel et al 1 elegantly illustrate this phenomenon occurring in the L-type voltage-gated Ca v 1.2 (Ca 2+ channel) of arterial myocytes. Channel clustering is identified as the mechanistic outcome of protein kinase A (PKA)-dependent phosphorylation of Ca v 1.2 at serine residue 1928 in the channel C terminus leading ultimately to enhanced Ca 2+ entry and vasoconstriction.…”

“…In this issue of Circulation Research , Baudel et al 1 apply an impressive battery of state-of-the-art imaging, electrophysiology, and biochemistry techniques to comprehensively elucidate the mechanism underlying Ca v 1.2 clustering and cooperative gating in arteriole smooth muscle cells (Figure). 1 Using a mouse model of diabetic hyperglycemia with documented alterations in Ca v 1.2 channel activity, the authors elegantly demonstrate how local AKAP5-dependent protein complexes 10 recruit PKA activity through the nucleotide stimulated G S -coupled P2Y 11 (purinergic receptor) resulting in the targeted phosphorylation of serine residue 1928 within the Ca v 1.2 C terminus (Figure). The resulting effect led to enhanced Ca 2+ entry and vasoconstriction, which could be eliminated by the phosphoablation of S1928 to alanine, the removal of AKAP5 (A-kinase anchoring protein 5), or the pharmacologic inhibition of PKA.…”

“…Importantly, although the study from Baudel et al 1 depicts clustering as a physiologic state necessary for proper timing and thresholding of signal generation, it also illustrates that coupled channel interactions can promote pathologic states, and that uncoupling can be an opportunity to mitigate disease. Indeed, pharmacologic blockers used to attenuate over-active channels are often met with limited success and often promote arrhythmias when exposed to the heart.…”

“…Although the new findings from the Baudel group 1 provide important insight into the mechanistic pathway linking posttranslational modification of Ca v 1.2, changes in channel clustering and function, they also raise a number of compelling unanswered questions. Given that functional channel coupling has been observed for many tissues where Ca v 1.2 is expressed, future directions will need to explore how universal this mechanism is throughout those tissues, as well as whether the other Ca v isoforms utilize a similar signaling network to modulate channel clustering and cell function.…”