Coupling of vascular CaV1.2 channels in health and disease

抽象的。 在健康和疾病中，给定的蛋白质如何组织成功能复合体尤其与 膜蛋白是细胞的关键信息入口点。一个合适且高度相关的例子 是电压门控的L型钙通道，在动脉肌细胞功能和血管中起主要作用 反应性。这些通道被证明是一致的(即协同门控)来放大钙离子的内流。在… 然而，目前对促进CaV1.2合作的诱导的机制有了全面的理解 门控及其在健康和疾病中的功能影响是主要的知识差距。整体而言 这项建议的目的是调查需求和生理后果， 血管CaV1.2通道C末端单一氨基酸S1928的磷酸化促进 CaV1.2的动态空间组织有助于表膜上的协作门控。要完成 为了达到这个目标，我们正在检验CaV1.2 S1928磷酸化调节动态通道的中心假设 聚集和协作门控，这有助于调节血管功能，以响应升高 细胞外葡萄糖和糖尿病期间。这一假设是在强有力和严谨的基础上提出的 初步数据显示S1928磷酸化作为疾病的罪魁祸首具有意想不到的显著作用 CaV1.2亚基在动脉肌细胞表面膜上的重新分布和组装成超簇 因为血糖升高和糖尿病。S1928磷酸化介导的CaV1.2超星团促进 CaV1.2协同门控和Ca~(2+)内流放大作用。主要发现进一步 强调我们观察到的重要性是CaV1.2 S1928磷酸化是必要的 高血糖时高血压信号通路的激活、血管收缩和血流改变 在糖尿病期间。此外，批判性的观察结果在新鲜分离的人类动脉中得到了验证。 来自非糖尿病和糖尿病患者的心肌细胞，强调了翻译的相关性。新兴市场和 将在本申请中探索的创新概念是S1928磷酸化作为1)a CaV1.2功能和血管反应性的变阻器以及2)血管并发症的主要危险因素 糖尿病。一种多尺度的当代方法，包括创新的显微技术，复杂的 将应用生物化学、电生理学、电子分析和独特的动物模型来探索 遵循目标。目的1阐明S1928磷酸化在动态CaV1.2聚集和 合作控制血糖升高。目标2是研究S1928磷酸化对 诱导糖尿病大鼠动脉肌细胞CaV1.2超聚集和协同门控。结果将发生变化 我们对CaV1.2如何在健康和健康的动脉肌细胞(可能还有其他细胞)中组织的理解 并可能为新的治疗策略奠定基础，以单一氨基酸的准确性纠正 通道功能和血管反应性。

Abstract . How a given protein organizes into a functional complex in health and disease is particularly relevant for membrane proteins that serve as key information entry points for cells. A suitable and highly relevant example is voltage-gated L-type CaV1.2 channels, which play a major role in arterial myocyte function and vascular reactivity. These channels have been shown to gate in unison (i.e. cooperative gating) to amplify Ca2+ influx. At present, however, a comprehensive understanding of mechanisms fostering the induction of CaV1.2 cooperative gating as well as its functional implications in health and disease represent major knowledge gaps. The overall objective of this proposal is to investigate the requirement and physiological consequences whereby phosphorylation of a single amino acid – S1928 – in the C-terminal of vascular CaV1.2 channels promotes dynamic spatial organization of CaV1.2 to facilitate cooperative gating at the surface membrane. To accomplish this goal, we are testing the central hypothesis that CaV1.2 S1928 phosphorylation tunes dynamic channel clustering and cooperative gating, and that this contributes to modulate vascular function in response to elevated extracellular glucose and during diabetes. This hypothesis is formulated on the basis of strong and rigorous preliminary data revealing an unanticipated and remarkable role for S1928 phosphorylation as the culprit for redistribution and assembly of CaV1.2 subunits into superclusters at the surface membrane of arterial myocytes upon elevated glucose and diabetes. CaV1.2 superclusters mediated by S1928 phosphorylation promotes CaV1.2 cooperative gating and Ca2+ influx amplification into arterial myocytes. Key findings that further underscore the significance of our observations is that CaV1.2 S1928 phosphorylation is necessary for activation of prohypertensive signaling pathways, vasoconstriction and altered blood flow upon elevated glucose and during diabetes. Moreover, critical observations have been validated in freshly dissociated human arterial myocytes from nondiabetic and diabetic patients, underscoring the translational relevance. Emerging and innovative concepts that will be explored in this application are the role of S1928 phosphorylation as 1) a rheostat of CaV1.2 function and vascular reactivity and 2) a major risk factor for vascular complications in diabetes. A multiscale contemporary approach that includes innovative microscopy techniques, sophisticated biochemistry, electrophysiology, in silico analysis and unique animal models will be implemented to explore the following aims. Aim 1 is to elucidate the role of S1928 phosphorylation in dynamic CaV1.2 clustering and cooperative gating upon elevated glucose. Aim 2 is to examine the requirement of S1928 phosphorylation to induce CaV1.2 superclustering and cooperative gating in arterial myocytes during diabetes. Results will transform our understanding of how CaV1.2 are organized in arterial myocytes (and perhaps other cells) in health and disease and may lay the foundation for novel therapeutic strategies with single amino acid accuracy to correct channel function and vascular reactivity.