Coupling of Vascular Cav1.2 Channels In Health & Disease

健康中血管 Cav1.2 通道的耦合

• 批准号: 8960054
• 负责人: Manuel F Navedo
• 金额: $ 54.61万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8960054
• 关键词: Address; Animal Model; Arteries; Binding Sites; Biosensor; Blood; Blood Pressure; Blood Vessels; Calcineurin; Calcium; Caliber; Calmodulin; Cardiovascular system; Cell physiology; Complex; Confocal Microscopy; Coupled; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diabetes Mellitus; Disease; Electrophysiology (science); Event; Figs - dietary; Fluorescence; Functional disorder; Genetic Transcription; Goals; Health; Human; Hypertension; Knowledge; Light; Membrane; Membrane Potentials; Modality; Modeling; Molecular Biology; Mus; Muscle Cells; Muscle function; Obesity; Optics; Organ; Outcome; Phosphotransferases; Physiological; Play; Process; Protein Kinase C Alpha; Proteins; Regulation; Reporter; Research; Resistance; Role; Route; Scaffolding Protein; Signal Pathway; Signal Transduction; Smooth Muscle; Source; Stroke; Surface; System; Tail; Telemetry; Testing; Time; Tissues; Total Internal Reflection Fluorescent; Vascular Diseases; Work; arteriole; base; constriction; diabetic; diabetic patient; hypertensive heart disease; innovation; insight; non-diabetic; novel; optogenetics; pandemic disease; public health relevance; research study; response; sensor; therapeutic target; tool; transcription factor; transcription factor NF-AT c3; vasoconstriction

 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.

 描述（由申请人提供）：血管 Cav1.2 通道是动脉肌细胞中 Ca2+ 进入的主要来源。因此，这些通道对于多种动脉功能发挥着关键作用，包括兴奋-收缩（EC）和兴奋-转录（ET）耦合。我们最近发现了 Cav1.2 通道的一种新门控模式，其中这些通道的一小部分可以一致门控（即耦合门控）。对这种门控方式或其在健康和疾病中调节动脉张力和血压的功能影响缺乏全面的机制理解 代表了知识上的重大差距。该提案旨在研究 Cav1.2 通道复合体中普遍存在的 Ca2+ 传感器和调节分子钙调蛋白 (CaM) 的动力学是这些通道之间耦合的基础的结构要求和生理后果。为了实现这一目标，我们正在测试新的中心假设，即 CaM 作为 Cav1.2 耦合调谐器，响应胞质 Ca2+ 的变化以及 PKA 和 PKCa 的激活，它们是调节动脉肌细胞和其他部位的 Cav1.2 的关键分子。在此模型中，AKAP150 充当锚定 PKA、PKCa 和钙调磷酸酶调节局部通道的枢纽，并充当促进相邻通道之间耦合的“纽带”。这一中心假设是在强有力的初步数据的基础上制定的，并将使用逻辑实验进程进行测试，该进程利用了 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分离 来自非糖尿病和糖尿病患者的完整人类动脉和动脉肌细胞。目标 1 将通过检查 CaM 促进 Cav1.2 耦合的结构要求来阐明 Cav1.2 通道耦合门控的机制。目标 2 将通过检查完整动脉中 CaM 动态、耦合事件和动脉肌细胞功能之间的关系来确定 Cav1.2 耦合在动脉肌细胞和完整动脉中的功能后果。目标 3 以前面的目标为基础，阐明 Cav1.2 与糖尿病期间动脉功能障碍耦合的重要性。我们将评估耦合事件、CaM 动力学的作用以及 AKAP150 协调的信号模块在糖尿病期间血管功能障碍发展中的作用。所提出的工作在技术层面上具有创新性，因为它能够揭示 Cav1.2 耦合的潜在机制，并且具有整合这种门控模式结果的独特能力，因为它与糖尿病期间血管并发症相关的现代定量框架中的 C 和 ET 耦合有关。这些结果将具有重要意义，因为它们将提供有关糖尿病期间 Cav1.2 通道耦合增加导致血管功能障碍的机制的新基本信息，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在来自非糖尿病和糖尿病患者的新鲜分离的人动脉肌细胞中得到验证，从而强调了我们应用的转化意义。