A single amino acid in CaV1.2 controls channel activity and arterial tone in hypertension

在动脉肌细胞中，钙离子通过电压门控的L型钙离子通道(LTCC)单通道或成簇内流 对血管反应性的主要调节影响，并最终影响生理和 病理情况，如高血压(HTN)。HTN的特点是血管紧张素II(AngII)增强 发信号。尽管大量证据表明，通过GQ/PKC轴作用的AngII刺激LTCC 活性、确切的激活机制、对血管反应性的影响以及与HTN的联系尚不清楚。 这项建议的总体目标是解决这一根本的知识差距。这里有初步数据， 为这些查询提供了一个独特的窗口，并揭示了单个动脉CaV1.2氨基酸作为 AngiI信号和HTN过程中LTCC功能和血管反应性增强的罪魁祸首。我们的预赛 数据表明，在增强的AngII信号转导过程中，LTCC活性和血管收缩增加是以下因素的结果 在S1928，PKC介导的CaV1.2磷酸化增加，这是一种假定的PKA磷酸化 与心脏血流动力学无功能相关性的部位。S1928磷酸化增强(PS1928)是 以前不受欢迎的CaV1.2亚基在肌膜中重新分布和组装成更大的簇， 将相邻的LTCC同步提升到栅极(即耦合栅极)。功能性pS1928介导的LTCC偶联 结果导致钙离子内流的净放大导致高血压信号通路的激活， 血管收缩和血流/压力改变在增强的AngII信号和HTN过程中，因此强调 我们数据的重要性。这些数据导致了新的中心假设的形成，即 动脉CaV1.2亚基中单个氨基酸S1928的磷酸化状态调节LTCC功能和 血管反应性在血管紧张素Ⅱ信号和HTN增强。超越了S1928作为一个 与功能相关的PKC磷酸化位点，一个新兴和创新的概念是pS1928是一个变阻器 LTCC功能和血管反应性，是HTN的主要危险因素。使用多尺度方法， 使用在我们团队中成熟的现代方法，包括创新的显微技术， 先进的生物化学、电生理学、遥测、电子分析和独特的动物模型，我们将 探索以下目标。目的1验证pS1928对血管紧张素转换酶依赖的增强必不可少的假设 LTCC活性和血管反应性。目的2检验pS1928促进LTCC增加的假设 增强的Angii信令期间的集群和耦合门控。最后，Aim 3测试了增强的假设 PS1928是导致CaV1.2团簇形成增加和LTCC活动导致 HTN中的血管功能障碍。结果将提供新的见解，将单个CaV1.2氨基酸与改变的 在AngiI信号和HTN增强过程中LTCC的功能和血管反应性，为新的 具有单一氨基酸准确性的治疗策略，以纠正LTCC活动和血管功能障碍。

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.