Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca_V1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca_V1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca_V1.2 channel pore-forming subunit (α1_C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca_V1.2 channel activity were associated with PKA activity, leading to α1_C phosphorylation at Ser¹⁹²⁸ Compared to arteries from low-fat diet (LFD)-fed mice and nondiabetic patients, arteries from high-fat diet (HFD)-fed mice and from diabetic patients had increased Ser¹⁹²⁸ phosphorylation and Ca_V1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser¹⁹²⁸ phosphorylation and Ca_V1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser¹⁹²⁸ phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca_V1.2 with Ser¹⁹²⁸ mutated to alanine (S1928A) lacked glucose-mediated increases in Ca_V1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca_V1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1_C phosphorylation at Ser¹⁹²⁸ in stimulating Ca_V1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes.

Large-conductance Ca²⁺-activated potassium (BK_Ca) channels are key determinants of vascular smooth muscle excitability. Impaired BK_Ca channel function through remodeling of BK_Ca β1 expression and function contributes to vascular complications in animal models of diabetes. Yet, whether similar alterations occur in native vascular smooth muscle from humans with type 2 diabetes is unclear. In this study, we evaluated BK_Ca function in vascular smooth muscle from small resistance adipose arteries of non-diabetic and clinically diagnosed type 2 diabetic patients. We found that BK_Ca channel activity opposes pressure-induced constriction in human small resistance adipose arteries, and this is compromised in arteries from diabetic patients. Consistent with impairment of BK_Ca channel function, the amplitude and frequency of spontaneous BK_Ca currents, but not Ca²⁺ sparks were lower in cells from diabetic patients. BK_Ca channels in diabetic cells exhibited reduced Ca²⁺ sensitivity, single-channel open probability and tamoxifen sensitivity. These effects were associated with decreased functional coupling between BK_Ca α and β1 subunits, but no change in total protein abundance. Overall, results suggest impairment in BK_Ca channel function in vascular smooth muscle from diabetic patients through unique mechanisms, which may contribute to vascular complications in humans with type 2 diabetes.

Background

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.

Methods

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.

Results

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 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 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.

Elevated glucose increases vascular reactivity by promoting L-type Ca_V1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a G_s-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y₁₁, the only G_s-coupled P2Y receptor, was detected in nanometer proximity to Ca_V1.2 and PKA. FRET-based experiments revealed that the selective P2Y₁₁ agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y₁₁ inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y₁₁-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y₁₁ in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.

The L-type Ca²⁺ channel Ca_V1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca²⁺ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y₁₁ agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca²⁺ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y₁₁/ P2Y₁₁-like receptors, AC5, PKA and Ca_V1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca²⁺ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia.