Coupling of Vascular Cav1.2 Channels In Health & Disease

 描述(申请人提供)：血管Cav1.2通道是动脉肌细胞内钙离子进入的主要来源。因此，这些通道在多种动脉功能中起着关键作用，包括兴奋-收缩(EC)和兴奋-转录(ET)耦合。我们最近确定了Cav1.2通道的一种新的选通方式，其中这些通道的一小部分可以同时选通(即耦合选通)。对这种门控方式或其在调节健康和疾病中的动脉张力和血压方面的功能影响缺乏全面的机械理解 代表着知识上的重大差距。该建议旨在研究Cav1.2通道复合体中普遍存在的钙传感器和调节分子钙调蛋白(CaM)的动力学支持这些通道之间的耦合的结构要求和生理后果。为了实现这一目标，我们正在测试一个新的中心假设，即CaM作为Cav1.2偶联调谐器来响应细胞内钙离子的变化，以及PKA和PKCA的激活，这两个关键分子调节动脉心肌细胞和其他地方的Cav1.2。在这个模型中，AKAP150通过锚定的PKA、PKCA和钙调神经磷酸酶作为局部通道调节的枢纽，并作为促进相邻通道之间耦合的“键”。这一中心假说建立在强大的初步数据基础上，并将利用合乎逻辑的实验进程进行测试，该实验进程利用PI或合作者实验室中成熟的方法，如异源表达系统、光遗传学、荧光生物传感器、分子生物学、电生理学、共聚焦和TIRF显微镜、遥测、糖尿病动物模型，以及从非糖尿病和糖尿病患者中分离完整的人类动脉和动脉肌细胞。目标1将通过研究CaM促进Cav1.2连接的结构要求来阐明Cav1.2通道耦合门的潜在机制。目的2通过研究CaM动力学、偶联事件与完整动脉肌细胞功能之间的关系，确定Cav1.2偶联在动脉和完整动脉中的功能后果。目标3建立在前面目标的基础上，目的是阐明Cav1.2偶联对糖尿病期间动脉功能障碍的重要性。我们将评估耦合事件、CaM动力学以及由AKAP150编排的信号模块在糖尿病血管功能障碍发展中的作用。这项拟议的工作在技术层面上是创新的，它揭示了Cav1.2偶联的潜在机制，并具有独特的能力，将这种门控模式的结果整合到与糖尿病期间血管并发症相关的现代定量框架中与C和ET偶联有关。这样的结果将是重要的，因为它们将提供关于Cav1.2通道偶联增加导致糖尿病期间血管功能障碍的机制的新的基本信息，并可能有助于开发治疗这种病理状况的合理疗法。重要的是，我们模型的关键概念已经在新分离的非糖尿病和糖尿病患者的人动脉肌细胞中得到验证，从而强调了我们的应用的翻译意义。

 DESCRIPTION (provided by applicant): Vascular Cav1.2 channels are the predominant source of Ca2+ entry in arterial myocytes. Consequently, these channels play a critical role for a wide variety of arterial functions, including excitation-contraction (EC) and excitation-transcription (ET) coupling. We have recently identified a new gating modality of Cav1.2 channels where a small subpopulation of these channels can gate in unison (i.e. coupled gating). The lack of a comprehensive mechanistic understanding of this gating modality or its functional implications in the regulation of arterial tone and blood pressure in health and disease represents major gaps in knowledge. This proposal aims to investigate the structural requirements and physiological consequences whereby dynamics of the ubiquitous Ca2+ sensor and regulatory molecule calmodulin (CaM) within the Cav1.2 channel complex underlies the coupling between these channels. To accomplish this, we are testing the novel central hypothesis that CaM serves as a Cav1.2 coupling tuner in response to changes in cytosolic Ca2+, and activation of PKA and PKCa, which are key molecules regulating Cav1.2 in arterial myocytes and elsewhere. In this model, AKAP150 serves as a hub for local channel regulation by anchored PKA, PKCa and calcineurin, and as the "bond" that facilitates coupling between adjacent channels. This central hypothesis has been formulated on the basis of strong preliminary data, and will be tested using a logical experimental progression that takes advantage of approaches well-established in the PI's or collaborators' labs such as heterologous expression systems, optogenetics, fluorescent biosensors, molecular biology, electrophysiology, confocal and TIRF microscopy, telemetry, animal models of diabetes, and isolation of intact human arteries and arterial myocytes from non-diabetic and diabetic patients. Aim 1 will elucidate the mechanisms underlying coupled gating of Cav1.2 channels by examining the structural requirements by which CaM promotes Cav1.2 coupling. Aim 2 will determine the functional consequences of Cav1.2 coupling in arterial myocytes and intact arteries by examining the relationship between CaM dynamics, coupled events and arterial myocyte function in intact arteries. Aim 3 builds on the preceding aims to elucidate the importance of Cav1.2 coupling to arterial dysfunction during diabetes. We will evaluate the role of coupled events, CaM dynamics and the contributions of the signaling module orchestrated by AKAP150 in the development of vascular dysfunction during diabetes. The proposed work is innovative at the technical level, in its ability to unmask underlying mechanisms of Cav1.2 coupling, and its unique ability to integrate the results of this gating modality as it relates to C and ET coupling in a modern quantitative framework that relates to vascular complications during diabetes. Such outcomes will be significant because they will provide new fundamental information on the mechanisms by which increased coupling of Cav1.2 channels underlies vascular dysfunction during diabetes and may contribute to the development of rational therapies for the treatment of this pathological condition. Importantly, critical concepts of our model have been validated in freshly dissociated human arterial myocytes from non- diabetic and diabetic patients, thus underscoring the translational significance of our application.