Ca2+ influx via single or clusters of voltage-gated L-type CaV1.2 channels (LTCC) in arterial myocytes exerts a major regulatory influence on vascular reactivity and ultimately on blood flow/pressure during physiological and pathological conditions such as hypertension (HTN). HTN is characterized by enhanced angiotensin II (angII) signaling. Although substantial evidence suggests that angII acting through the Gq/PKC axis stimulates LTCC activity, the precise activation mechanism, its impact on vascular reactivity and the link to HTN are unknown. The overall objective of this proposal is to address this fundamental knowledge gap. Preliminary data herein, offer a unique window into these queries and uncover an essential role of a single arterial CaV1.2 amino acid as the culprit for enhanced LTCC function and vascular reactivity during angII signaling and HTN. Our preliminary data indicate that elevated LTCC activity and vasoconstriction during enhanced angII signaling is the result of increased PKC-mediated CaV1.2 phosphorylation at S1928, which remarkably is a putative PKA phosphorylation site with no functional relevance in heart hemodynamics. Increased S1928 phosphorylation (pS1928) underlies a previously unappreciated redistribution and assembly of CaV1.2 subunits into larger clusters in the sarcolemma, promoting adjacent LTCCs to gate in unison (i.e. coupled gating). Functional pS1928-mediated LTCC coupling results in a net amplification of Ca2+ influx leading to activation of prohypertensive signaling pathways, vasoconstriction and altered blood flow/pressure during enhanced angII signaling and HTN, thus underscoring the significance of our data. These data led to the formulation of the novel central hypothesis that the phosphorylation state of a single amino acid - S1928 - in the arterial CaV1.2 subunit, tunes LTCC function and vascular reactivity during enhanced angII signaling and HTN. Beyond the unforeseen role for S1928 as a functionally relevant PKC phosphorylation site, an emerging and innovative concept is that pS1928 is a rheostat of LTCC function and vascular reactivity, and a major risk factor for HTN. Using a multiscale approach that employs contemporary methods well-established in our group, including innovative microscopy techniques, sophisticated biochemistry, electrophysiology, telemetry, in silico analysis, and unique animal models, we will explore the following aims.
Aim 1 tests the hypothesis that pS1928 is essential for angII-dependent augmentation of LTCC activity and vascular reactivity.
Aim 2 test the hypothesis that pS1928 facilitates increased LTCC clustering and coupled gating during enhanced angII signaling. Finally, Aim 3 tests the hypothesis that enhanced pS1928 is the principal mechanism underlying increased CaV1.2 cluster formation and LTCC activity leading to vascular dysfunction in HTN. Outcomes will offer new insight linking a single CaV1.2 amino acid with altered LTCC function and vascular reactivity during enhanced angII signaling and HTN and lay the foundation for novel therapeutic strategies with single amino acid accuracy to correct LTCC activity and vascular dysfunction.
Arterial myocytes wrapping around the wall of blood vessels control vascular function and are essential for proper regulation of blood pressure. Alteration in mechanisms regulating arterial myocyte contractility may lead to high blood pressure (i.e. hypertension). Hypertension is a devastating disease affecting millions of Americans with costs over $50 billion/year, and a major risk factor for several cardiovascular complications, including stroke, peripheral vascular disease, and cerebral small vessel diseases. Here we will carry out careful quantitative studies to provide insight into novel molecular mechanisms impairing the function of a key protein regulating the contractility of arterial myocytes and therefore vascular function and blood pressure during hypertension. The results from the proposed work will fulfill relevant public health goals and NIH mission by generating fundamental knowledge that could identify valuable therapeutic targets, as well as to help develop novel treatment strategies for cardiovascular complications, including hypertension.
