Mechanisms of Long-term Cardiac Ion Channel Regulation

描述（由申请人提供）：心力衰竭（HF）和房颤（AF）是美国最常见的两种心脏病。在中度至重度HF和房颤中，会发生许多表型变化，包括电压门控钙通道电流密度降低。由于Ca通道在兴奋收缩耦合中起中心作用，其表达的变化可能对心脏功能产生深远的影响。此外，Ca通道也参与了激发-转录耦合。因此，心脏Ca通道表达的改变也可以影响疾病的进展。心脏主电压门控钙通道（CaV1.2）在其生物物理特性和第二信使调制领域被广泛研究。然而，关于Ca通道表达的长期调节的信息非常稀疏。在本研究中，我们提出了一种新的机制假说来控制心肌细胞肌膜中Ca通道的功能表达。在我们的初步数据中，我们显示了ras相关的单体g蛋白与CaV1.2辅助亚基CaVb2相互作用的证据。我们建议验证g蛋白与CaVb亚基的相互作用通过竞争CaV1.2结合来降低Ca通道电流密度的全局假设。此外，我们的初步数据显示了强有力的证据，表明这是一个动态过程，是由慢性PKA活动调节的。有四个具体目标指导我们的实验设计：目标1将表征新生g蛋白和CaVb复合物的时间过程和亚细胞定位。目的2将描述细胞内第二信使g蛋白- cavb对Ca通道电流表达的调节。目的3将描述Ca通道电流和g蛋白在天然细胞中的调节。目的4将描述这些g蛋白在心脏功能中的功能。这些目标涵盖了从纯分子性质（蛋白质-蛋白质相互作用）的研究，到体外模型系统（重组蛋白的异种表达），再到天然细胞系统（心脏细胞的原代分离和培养），再到单一类g蛋白对组织水平心脏功能的影响。这一建议可能会确定一类新的治疗靶点，以减轻心衰和房颤的心功能障碍。

DESCRIPTION (provided by applicant): Heart failure (HF) and atrial fibrillation (AF) represent two of the most prevalent heart diseases in the U.S. In moderate to severe HF and in AF numerous phenotypic changes occur including a reduction of voltage-gated calcium channel current density. Changes in Ca channel expression may have far-reaching effects on heart function due to their central role in excitation contraction coupling. Also, Ca channels have been implicated in excitation-transcription coupling. Thus alterations of Ca channel expression in the heart can also impact on disease progression. The principal voltage-gated Ca channel in the heart (CaV1.2) is extensively studied in the realm of its biophysical characteristics and second messenger modulation. However, very sparse information is available regarding longer term regulation of Ca channel expression. In this proposal we introduce a novel mechanistic hypothesis governing the functional expression of cardiac Ca channels in the sarcolemma of cardiac myocytes. In our preliminary data we show evidence for a ras-related monomeric G-protein interaction with the CaV1.2 accessory subunit CaVb2. We propose to test the global hypothesis that G-protein interaction with CaVb subunits reduces Ca channel current density by competing for CaV1.2 binding. Moreover, our preliminary data shows strong evidence that this is a dynamic process that is modulated by chronic PKA activity. There are four specific aims that guide our experimental design: Aim 1 will characterize the time course and sub-cellular localization of nascent G-protein and CaVb complexes. Aim 2 will characterize the intracellular second messenger modulation of G-protein-CaVb regulation of Ca channel current expression. Aim 3 will characterize the modulation of Ca channel current and G-protein modulation in native cells. Aim 4 will characterize the function of these G-proteins in heart function. These aims encompass approaches ranging from investigations of a purely molecular nature (protein-protein interactions), to in vitro model systems (heterologous expression of recombinant proteins), to native cell systems (primary isolates and cultures of heart cells), to the impact of a single class of G-proteins on tissue level heart function. This proposal may identify a novel class of therapeutic targets for alleviation of heart dysfunction in HF and AF.