Functional roles of atrial-specific ion channels in the heart

项目总结/摘要 心房颤动（AF）是影响退伍军人群体的最常见的房性心律失常， 有很大的栓塞和中风风险这个问题进一步恶化的事实是， 事实证明，这些战略在很大程度上是不够的。在上一次融资期间，我们发现了令人惊讶的， 具有广泛治疗意义的深刻发现。与先前的报告相比， Cav1.3（a1 D）L型钙通道（LTCC）主要表达于神经元和神经内分泌细胞，我们发现， Cav 1.3钙通道在心房肌细胞中有明显表达。事实上，Cav1.3 Ca 2+通道 与心室肌细胞相比，优先在心房中表达。此外，Cav1.3 Ca 2+的重要性 心房中的通道是由通道的空删除导致显著的 心房兴奋性改变、房性心律失常以及深窦房（SA）和房室（AV） 淋巴结功能障碍我们进一步证明Cav1.2和Cav1.3通道形成多聚体蛋白， 复合物与小电导钙激活的K+通道（SK通道）的心脏，这是第一次 最近在我们的实验室里发现的。主要的有趣和功能的重要性是发现，SK 通道也优先在心房肌细胞以及心脏的起搏组织中表达。我们 进一步证明SK2通道通过物理桥与Cav1.3和Cav1.2相关， 肌动蛋白2。此外，我们还获得了新的初步数据，这些数据表明， 细胞骨架蛋白在SK2通道的适当膜定位中是关键的。鉴于这些相关 数据，我们将直接检查SK通道贩运的分子决定因素。此外，功能 将直接描述新近描述的SK通道在起搏组织中的作用。我们的研究结果 代表了一个统一的分子和细胞机制的开始， 心房肌细胞Cav通道和Ca 2+激活的K+通道（KCa）之间的时空串扰。嵌入式 在这些发现中，相关的范式转变可能被用于开发心房特异性药物， 治疗房性心律失常。因此，该提案的总体主旨是部署新的分子和 功能策略，许多灵感来自渠道机制的研究，为发现的基本和 心脏中Ca 2+和K+通道的新可及区域。 我们将使用体外相互作用测定、共聚焦显微镜和免疫荧光法的组合来直接检验这一假设。 成像、电子显微镜分析、基因沉默、生物化学研究和功能分析。最后， 我们假设SK通道的所有3种亚型都在SA和AV结细胞中表达， 关键在于节点细胞的放电和动作电位持续时间。我们建议直接检验假设 使用SK 1、SK2和SK 3通道的无效突变模型。我们建议的研究将大大扩大我们的 了解单个Ca 2+和SK通道的具体功能。事实上， 心房特异性和起搏组织特异性离子通道可提供靶向这些通道的新方法 而不干扰心室组织的兴奋性。

Project Summary/Abstract Atrial fibrillation (AF) is the most common atrial arrhythmia affecting veteran population, and is associated with a significant risk of embolism and stroke. The problem is further exacerbated by the fact that treatment strategies have proven largely inadequate. During the last funding period, we uncovered surprising, yet insightful findings that have broad therapeutic ramifications. In contrast to previous reports that suggested that Cav1.3 (a1D) L-type Ca2+ channel (LTCC) is expressed mainly in neurons and neuroendocrine cells, we demonstrated significant expression of Cav1.3 Ca2+ channel in atrial myocytes. Indeed, Cav1.3 Ca2+ channel is preferentially expressed in atrial compared to ventricular myocytes. Additionally, the importance of Cav1.3 Ca2+ channels in atria is underpinned by the revelation that null deletion of the channel results in significant alteration in atrial excitability, atrial arrhythmias as well as profound sinoatrial (SA) and atrioventricular (AV) nodes dysfunction. We further demonstrated that Cav1.2 and Cav1.3 channels form multimeric protein complexes with small conductance Ca2+-activated K+ channels (SK channels) in the heart, which were first uncovered recently in our laboratory. Of major intriguing and functional importance are the findings that SK channels are also preferentially expressed in atrial myocytes as well as pacemaking tissues of the heart. We further demonstrated that SK2 channels associate with Cav1.3 and Cav1.2 through a physical bridge, a- actinin2 in cardiac myocytes. In addition, we have obtained new preliminary data, which demonstrate that cytoskeletal proteins are critical in the proper membrane localization of SK2 channel. Given these relevant data, we will directly examine the molecular determinants of SK channel trafficking. Moreover, the functional roles of the newly described SK channels in pacemaking tissues will be directly delineated. Our findings represent the beginning of a unified molecular and cellular mechanism that demonstrates a functional spatiotemporal cross talk between Cav channels and Ca2+-activated K+ channels (KCa) in atrial cells. Embedded in these findings are relevant paradigm shifts that may be exploited in developing atrial-specific drugs for the treatment of atrial arrhythmia. Hence, the overall thrust of the proposal is to deploy new molecular and functional strategies, many inspired from channel mechanistic studies, for the discovery of fundamental and newly accessible arenas of Ca2+ and K+ channels in the heart. We will directly test the hypothesis using a combination of in vitro interaction assay, confocal microscopic imaging, electron microscopic analyses, gene silencing, biochemical studies and functional analyses. Finally, we hypothesize that all 3 isoforms of SK channels are expressed in SA and AV node cells and contribute critically to the firing and action potential durations of nodal cells. We propose to directly test the hypothesis using null mutant models of SK1, SK2 and SK3 channels. Our proposed studies will substantially expand our understanding of the specific functions of individual Ca2+ and SK channels. Indeed, novel insights into the atrial-specific and pacemaking tissue-specific ion channels may provide new means to target these channels without interfering with the excitability of ventricular tissues.