Ion channel regulation by macromolecular complexes

描述（由申请人提供）：通过大分子信号传导复合物和磷酸化对离子通道的调节在兴奋-收缩（E-C）偶联的调节中起重要作用。在心肌细胞中，L-型Ca 2+通道（Cav1.2）电流（ICa）是导致动作电位平台和E-C偶联的主要去极化电流，它在很大程度上通过蛋白激酶和磷酸酶的激活受到激素的高度调节。已经确定Cav1.2在响应于经典信号传导途径（例如，肾素-血管紧张素系统（RAS）和交感神经系统（SNS））调节心脏功能中起关键作用。几种主要心血管疾病的治疗部分依赖于药物对这些途径的调节。该提案的重点是更好地了解主要心脏蛋白激酶，蛋白激酶C（PKC），由RAS和SNS激活，调节Cav1.2功能的机制。我们绘制了Cav1.2 α 1c和β 2亚基上的关键PKC磷酸化位点，并制备了磷酸肽特异性抗体;每个抗体都被独特地设计为仅识别单个磷酸化Cav1.2通道位点。我们的初步数据表明，不同的心脏PKC亚型可以磷酸化不同的Cav1.2网站在体外。利用磷酸肽和位点特异性抗体，申请人提出研究不同PKC同种型在正常和病理心脏中Cav1.2调节中的作用。本研究的主要目的是：（1）研究Cav1.2在细胞、组织和动物中的磷酸化特征;（2）研究心肌Cav1.2的电生理PKC调节;（3）研究心肌肥厚和心力衰竭时Cav1.2的PKC调节。利用生物化学，分子生物学和电生理技术，我们将探索在正常和患病的心脏Cav1.2调节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进行特定Cav1.2残基的PKC磷酸化作用的电生理学表征。由于心力衰竭和肥大与PKC活性和Ca2+稳态的改变有关，因此了解Cav1.2的PKC调节可能有助于开发治疗这些疾病的新型治疗药物。

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.