Ion channel regulation by macromolecular complexes

大分子复合物对离子通道的调节

• 批准号: 7822261
• 负责人: Steven O Marx
• 金额: $ 1.45万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822261
• 关键词: 1,2-diacylglycerol; Action Potentials; Address; Adrenergic Receptor; Agonist; Angiotensin II; Animal Model; Antibodies; Arrhythmia; Biochemical; Biological; Calcium; Canis familiaris; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell physiology; Cells; Complex; Congestive Heart Failure; Coupling; Data; Development; Diglycerides; Dihydropyridines; Disease; Endothelin; Event; Family; Gene Expression; Heart; Heart Hypertrophy; Heart failure; Homeostasis; Hormones; Human; Hypertension; Hypertrophy; In Vitro; Ion Channel; Knock-in Mouse; Lead; Macromolecular Complexes; Maps; Mediating; Modeling; Molecular; Morphology; Mus; Neurotransmitters; Nodal; Pacemakers; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Phorbol Esters; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Property; Protein Isoforms; Protein Kinase; Protein Kinase C; Pump; Rattus; Reagent; Regulation; Relaxation; Renin-Angiotensin System; Research Personnel; Role; Sarcolemma; Signal Pathway; Signal Transduction; Site; Sympathetic Nervous System; System; Tachycardia; Techniques; Testing; Therapeutic Agents; Time; Tissues; Transfection; Upper arm; analog; animal tissue; base; depressed; design; dihydropyridine; insight; mutant; novel; novel therapeutics; pressure; programs; research study; response; translational study; voltage

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.

描述（由申请人提供）：大分子信号复合物和磷酸化对离子通道的调节在兴奋-收缩（E-C）耦合的调节中起着重要作用。在心肌细胞中，L 型 Ca2+ 通道 (Cav1.2) 电流 (ICa) 是导致动作电位和 E-C 耦合稳定的主要去极化电流，受到激素的高度调节，很大程度上是通过激活蛋白质激酶和磷酸酶。众所周知，Cav1.2 在响应经典信号通路（如肾素-血管紧张素系统 (RAS) 和交感神经系统 (SNS)）调节心脏功能方面发挥着关键作用。几种主要心血管疾病的治疗部分取决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。我们在Cav1.2 α1c和β2亚基上绘制了关键的PKC磷酸化位点，并制备了磷酸化肽特异性抗体；每个都经过独特设计，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 亚型可以在体外磷酸化不同的 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PKC亚型在正常和病理心脏中Cav1.2调节中的作用。提出了三个具体目标：（1）从生化和药理学角度表征细胞、组织和动物中Cav1.2的磷酸化； (2) 表征心脏 Cav1.2 的电生理学 PKC 调节； (3) 表征心力衰竭和肥厚中 Cav1.2 的 PKC 调节。利用生物化学、分子生物学和电生理学技术，我们将探索影响正常和患病心脏中 Cav1.2 调节的分子机制和细胞信号传导途径。将在异源表达系统和心肌细胞中对特定 Cav1.2 残基的 PKC 磷酸化影响进行电生理学表征。由于心力衰竭和肥厚与 PKC 活性和 Ca2+ 稳态的改变有关，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。