Ion channel regulation by macromolecular complexes

描述(申请人提供)：大分子信号复合体和磷酸化对离子通道的调节在兴奋-收缩(E-C)偶联的调节中起着重要作用。在心肌细胞中，L钙通道(Cav1.2)电流(ICa)是导致动作电位平台化和E-C偶联的主要去极化电流，它受激素的高度调节，在很大程度上是通过蛋白激酶和磷酸酶的激活来实现的。众所周知，Cav1.2在肾素-血管紧张素系统(RAS)和交感神经系统(SNS)等经典信号通路的调控中起着关键作用。几种主要心血管疾病的治疗在一定程度上取决于药物对这些通路的调节。这一建议的重点是更好地了解由RAS和SNS激活的主要心脏蛋白激酶--蛋白激酶C(PKC)调节Cav1.2功能的机制。我们已经绘制了Cav1.2α1c和β2亚基上关键的PKC磷酸化位点的图谱，并制备了磷酸化多肽特异性抗体；每个抗体都独特地设计为仅识别单个磷酸化的Cav1.2通道位点。我们的初步数据表明，在体外，不同的心脏PKC亚型可以磷酸化不同的Cav1.2位点。利用磷酸肽和位点特异性抗体，申请人提议研究不同的PKC亚型在正常和病理心脏中对Cav1.2的调节中的作用。我们提出了三个具体的目标：(1)从生化和药理学的角度研究Cav1.2在细胞、组织和动物中的磷酸化；(2)从电生理的角度研究Cav1.2对心脏Cav1.2的PKC调节；(3)研究Cav1.2在心力衰竭和肥厚中的PKC调节作用。利用生化、分子生物学和电生理学技术，我们将探索在正常和疾病心脏中影响Cav1.2调控的分子机制和细胞信号通路。将在异源表达系统和心肌细胞中对特定Cav1.2残基的PKC磷酸化的影响进行电生理学表征。由于心力衰竭和肥厚与PKC活性和钙稳态的改变有关，了解Cav1.2对PKC的调节可能有助于开发新的治疗药物来治疗这些疾病。

DESCRIPTION (provided by applicant): Modulation of ion channels by macromolecular signaling complexes and phosphorylation plays an important role in the regulation of excitation-contraction (E-C) coupling. In cardiac myocytes, the L-type Ca2+ channel (Cav1.2) current (ICa), the major depolarizing current contributing to the plateau of the action potential and E-C coupling, is highly regulated by hormones, in large part through the activation of protein kinases and phosphatases. It is well established that Cav1.2 plays a key role in modulating cardiac function in response to classical signaling pathways, such as the renin-angiotensin system (RAS) and sympathetic nervous system (SNS). Treatment of several major cardiovascular diseases is dependent, in part, upon the modulation of these pathways by drugs. This proposal focuses on gaining a better understanding of the mechanisms by which a major cardiac protein kinase, protein kinase C (PKC), which is activated by the RAS and SNS, modulates the function of Cav1.2. We have mapped critical PKC phosphorylation sites on the Cav1.2 alpha1c and beta2 subunits and have prepared phospho-peptide specific antibodies; each uniquely designed to recognize only a single phosphorylated Cav1.2 channel site. Our preliminary data suggest that the different cardiac PKC isoforms can phosphorylate distinct Cav1.2 sites in vitro. Utilizing the phospho-peptide and site specific antibodies, the applicant proposes to study the role of different PKC isoforms in the modulation of Cav1.2 in normal and pathological hearts. Three specific aims are proposed: (1) To characterize biochemically and pharmacologically the phosphorylation of Cav1.2 in cells, tissues and animals; (2) To characterize electrophysiologically PKC modulation of cardiac Cav1.2; (3) To characterize the PKC modulation of Cav1.2 in heart failure and hypertrophy. Utilizing biochemical, molecular biological, and electrophysiological techniques, we will explore the molecular mechanisms and cellular signaling pathways impinging upon Cav1.2 regulation in normal and diseased hearts. Electrophysiological characterization of the effects of PKC phosphorylation of specific Cav1.2 residues will be carried out in heterologous expression systems and cardiomyocytes. Since heart failure and hypertrophy are associated with alterations in PKC activity and Ca2+ homeostasis, understanding the PKC modulation of Cav1.2 may contribute to the development of novel therapeutic agents to treat these disorders.