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

描述(申请人提供)：心肌肥大最初是一种增加心输出量的代偿性反应。然而，持续的肥厚会导致扩张型心肌病和心力衰竭(HF)，这在美国每年导致超过26.2万人死亡。最近的研究表明，细胞内钙离子的病理性升高是触发心肌肥大基因程序的主要原因。有趣的是，瞬时外向K+通道(ITO)的阻断或基因缺失能够触发细胞生长和心肌肥大。综上所述，Ito可能参与心肌细胞内[Ca~(2+)]i的功能调节。我们最近报道，阻断Ito可显著促进小鼠心室肌细胞L型钙通道电流(ICa)的发生，我们推测Ito与ICa之间存在功能联系，钙/钙调素依赖的激酶II(CaMKII)参与了Ito与ICa之间的调控信号转导。在这里，我们扩展了我们的工作来检验这些假设。我们的第一个目标是通过免疫共沉淀和免疫细胞化学标记技术来测试Ito通道和CaMKII是否形成大分子复合体。第二个目的是测试CaMKII-Ito通道复合体是否响应Ito阻断而动态发挥作用(即CaMKII是否会被Ito通道阻滞剂取代)。第三个目的是测试ICA易化是否是被Ito通道阻滞剂取代了CaMKII-Ito通道复合体的CaMKII对钙通道磷酸化的结果。电生理学方法(全细胞膜片钳)和遗传学方法(CaMKII d基因敲除小鼠模型)将被用来检验这些假说。这里提出的概念是，Ito通道是CaMKII的储备库，而Ito通道阻滞剂对CaMKII的置换增加了可用来促进ICA的CaMKII。虽然由于早期和概念性阶段，预计风险很高，但我们最近发表的电生理学结果和来自其他组的免疫共沉淀数据强烈表明了这种可行性。检验所提出的假设可能会导致一个重要的、新的概念框架，并在一个新的研究领域取得突破。例如，ICA和Ito的功能关联在心肌肥厚和衰竭的发展中的作用将是直接感兴趣的领域。在美国，每年有超过26.2万人死于心肌肥厚和随之而来的心力衰竭。这一探索性的建议集中在膜K+电流(瞬时外向钾电流，ITO)上，这种电流在心力衰竭中一直被发现是一种继发性变化。我们的总体目标是测试阻断这一膜离子通道是否通过对心肌细胞钙通道(L型钙通道)的功能调节来促进心肌细胞内钙内流，并探索潜在的机制，这可能导致一个重要的新的概念框架，其中Ito的下调可能在促进心衰进程中发挥重要作用。

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.