Physical and functional coupling between transient outward K+ channel and CaMKII

瞬态外向 K 通道与 CaMKII 之间的物理和功能耦合

• 批准号: 7500373
• 负责人: YANGGAN WANG
• 金额: $ 23.55万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7500373
• 关键词: Aftercare; Antibodies; Area; Binding; Calcium; Calmodulin; Cardiac; Cardiac Myocytes; Cardiac Output; Cells; Co-Immunoprecipitations; Complex; Coupled; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dilated Cardiomyopathy; Dissociation; Down-Regulation; Electrophysiology (science); Failure; Genes; Genetic; Goals; Heart Hypertrophy; Heart failure; Hypertrophy; Immunoblotting; Ion Channel; Knock-out; Kv4.3 channel; L-Type Calcium Channels; Label; Lead; Life; Localized; Macromolecular Complexes; Membrane; Methods; Mus; Muscle Cells; Pattern; Peptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Potassium Channel; Process; Proteins; Publishing; Regulation; Reporting; Research; Risk; Role; Signal Transduction; Staging; Techniques; Testing; United States; Ventricular; Work; autocamtide-2; calmodulin-dependent protein kinase II; cell growth; channel blockers; concept; immunocytochemistry; interest; killings; mouse model; patch clamp; programs; research study; response

DESCRIPTION (provided by applicant): Cardiac hypertrophy is initially a compensatory response that augments cardiac output. However, sustained hypertrophy leads to dilated cardiomyopathy and heart failure (HF), which kills more than 262,000 lives a year in United States. Recent studies have demonstrated that the pathological increase of intracellular calcium [Ca2+]i is the primary cause of triggering cardiac hypertrophic gene program. Interestingly, blocking or genetic deletion of transient outward K+ channel (Ito) is capable of triggering cell growth and cardiac hypertrophy. Taken together, Ito may participate functional regulation of [Ca2+]i in cardiac myocytes. We recently reported that blocking of Ito significantly facilitates L-type calcium channel current (ICa) in mouse ventricular myocytes and we hypothesize that there is a functional association between Ito and ICa and that the Ca2+/Calmodulin- dependent kinase II (CaMKII) is involved in the transduction of regulatory signals between Ito and ICa. Here, we extend our work to test these hypotheses. Our first aim is to test whether Ito channel and CaMKII form a macromolecular complex by using techniques of co-immunoprecipitation and immunocytochemistry labeling. The second aim is to test whether CaMKII- Ito channel complex is dynamically functioning in response to Ito blocking (i.e. whether CaMKII will be displaced by Ito channel blocker). The third aim is to test whether ICa facilitation is the result of Ca2+ channel phosphorylation by CaMKII that displaced from CaMKII-Ito channel complex by Ito channel blocker. The electrophysiological (whole-cell patch clamp) and genetic (CaMKII d knockout mouse model) approaches will be employed to test these hypotheses. The proposed concept here is that Ito channel is a reservoir for CaMKII and the displacement of CaMKII by Ito channel blocker increases CaMKII available to facilitate ICa. Although high risk is anticipated due to the early and conceptual stage, the feasibility is strongly suggested by our recently published electrophysiology results and the co-immunoprecipitation data from other group. Testing the proposed hypothesis may lead to a significant and new conceptual framework and a breakthrough in a new area of research. For instance, the role of functional association of ICa and Ito in the development of cardiac hypertrophy and failure will be the immediate area of interest. Cardiac hypertrophy and the consequent heart failure kill more than 262,000 lives a year in United States. This exploratory proposal is focused on a membrane K+ current (transient outward K+ current, Ito) which is consistently found to be decreased in heart failure as a secondary change. Our overall goal is to test whether blocking this membrane ion channel facilitates Ca2+ influx of cardiomyocytes via functional regulation of the cardiac Ca2+ channel (L-type Ca2+ channel) and to explore the underlying mechanisms which may lead to a significant and new conceptual framework of which the down-regulation of Ito may play an important role in promoting the progression of HF process.

描述（由申请人提供）：心脏肥大最初是一种增加心输出量的代偿反应。然而，持续的肥大导致扩张型心肌病和心力衰竭（HF），在美国每年导致超过262，000人死亡。近年来的研究表明，心肌细胞内钙离子（[Ca 2 +]i）的病理性升高是触发心肌肥厚基因程序的主要原因。有趣的是，瞬时外向钾通道（Ito）的阻断或基因缺失能够触发细胞生长和心肌肥大。Ito可能参与了心肌细胞[Ca ~（2+）]i的功能调节。我们最近报道了阻断Ito显著促进小鼠心室肌细胞L型钙通道电流（伊卡），我们推测Ito和伊卡之间存在功能关联，并且Ca 2 +/钙调蛋白依赖性激酶II（CaMKII）参与Ito和伊卡之间的调节信号转导。在这里，我们扩展我们的工作来测试这些假设。我们的第一个目的是测试是否Ito通道和CaMK Ⅱ形成一个大分子复合物，通过使用免疫共沉淀和免疫细胞化学标记技术。第二个目的是测试CaMKII- Ito通道复合物是否响应于Ito阻断而动态地发挥功能（即CaMKII是否将被Ito通道阻断剂置换）。第三个目的是检测Ito通道阻断剂是否将CaMKII从CaMKII-Ito通道复合物中置换出来，使Ca 2+通道磷酸化，从而使伊卡易化。将采用电生理学（全细胞膜片钳）和遗传学（CaMKII d敲除小鼠模型）方法来检验这些假设。这里提出的概念是Ito通道是CaMKII的储存库，Ito通道阻断剂对CaMKII的置换增加了可用于促进伊卡的CaMKII。虽然由于早期和概念阶段，预计高风险，但我们最近发表的电生理结果和其他组的免疫共沉淀数据强烈建议了可行性。检验所提出的假设可能会导致一个重要的和新的概念框架，并在一个新的研究领域的突破。例如，伊卡和Ito在心脏肥大和衰竭发展中的功能关联的作用将是直接感兴趣的领域。在美国，心脏肥大和随之而来的心力衰竭每年导致超过262，000人死亡。这项探索性建议的重点是膜K+电流（瞬时外向K+电流，Ito），这是一致发现在心力衰竭作为继发性变化减少。我们的总体目标是测试是否通过心脏Ca 2+通道（L型Ca 2+通道）的功能调节来阻断该膜离子通道促进心肌细胞的Ca 2+内流，并探索可能导致一个重要的和新的概念框架的潜在机制，其中Ito的下调可能在促进HF过程中发挥重要作用。