Mechanisms of Long-term Cardiac Ion Channel Regulation

描述（由申请人提供）：心力衰竭（HF）和心房颤动（AF）是美国最常见的两种心脏病。在中度至重度HF和AF中，发生许多表型变化，包括电压门控钙通道电流密度降低。Ca通道表达的变化可能对心脏功能产生深远的影响，因为它们在兴奋收缩偶联中起着重要作用。此外，Ca通道与兴奋-转录偶联有关。因此，心脏中Ca通道表达的改变也可以影响疾病进展。心脏中的主要电压门控Ca通道（CaV1.2）在其生物物理特性和第二信使调节领域中被广泛研究。然而，关于Ca通道表达的长期调节的信息非常稀少。在这个建议中，我们介绍了一种新的机制假说，管理心肌细胞的肌膜中的心脏钙通道的功能表达。在我们的初步数据中，我们显示了与CaV1.2辅助亚基CaVb 2的Ras相关单体G蛋白相互作用的证据。我们建议测试的全球性假设，G蛋白与CaVb亚基的相互作用降低钙通道电流密度竞争CaV1.2结合。此外，我们的初步数据显示了强有力的证据，这是一个动态的过程，是由慢性PKA活性调制。有四个具体的目标，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将表征细胞内第二信使G蛋白-CaVb调节钙通道电流表达的作用。目的3将表征天然细胞中钙通道电流的调节和G蛋白的调节。目的4将描述这些G蛋白在心脏功能中的功能。这些目标包括从纯分子性质（蛋白质-蛋白质相互作用）的研究到体外模型系统（重组蛋白的异源表达），到天然细胞系统（心脏细胞的原代分离物和培养物），再到单一类别的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种新的治疗目标，以减轻心脏功能障碍的HF和AF。

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.