Functional roles of atrial-specific ion channels in the heart

项目摘要/摘要 房颤是影响退伍军人人群的最常见的房性心律失常，与 有很大的血栓和中风风险。这一问题进一步恶化，因为治疗 事实证明，这些战略在很大程度上是不够的。在上一个资助期，我们发现了令人惊讶的，但 有洞察力的发现，具有广泛的治疗后果。与之前的报告不同的是， Cav1.3(A1D)L型钙通道主要表达于神经元和神经内分泌细胞。 Cav1.3钙通道在心房肌细胞中有显著表达。事实上，Cav1.3钙通道是 与心室肌细胞相比，心房肌细胞优先表达。此外，Cav1.3钙离子的重要性 心房中的通道得到了如下发现的支持：通道的零删除会导致显著的 心房兴奋性、房性心律失常及深静脉窦房结和房室传导速度的改变 结节功能障碍。我们进一步证明了Cav1.2和Cav1.3通道形成多聚体蛋白 小电导钙激活钾通道(SK通道)的复合体，这是首次发现的 最近在我们实验室发现的。主要耐人寻味和功能重要性的是SK的发现 通道也优先在心脏的心房肌细胞和起搏组织中表达。我们 进一步证明了SK2通道通过物理网桥与Cav1.3和Cav1.2相关联，a- 心肌细胞中的肌动蛋白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通道膜的正确定位中起关键作用。鉴于这些相关内容 数据，我们将直接考察SK渠道交易的分子决定因素。此外，功能 新描述的SK通道在起搏组织中的作用将被直接描述。我们的发现 代表了一个统一的分子和细胞机制的开始，它展示了一种功能 心房细胞Cav通道和钙激活钾通道(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利用来开发针对心房的特异性药物 房性心律失常的治疗。因此，该提案的总体主旨是部署新的分子和 功能性策略，其中许多是从渠道机制研究中获得的，用于发现基础和 心脏内新进入的钙通道和钾通道的区域。 我们将使用体外相互作用分析、共聚焦显微镜 成像、电子显微镜分析、基因沉默、生化研究和功能分析。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，并参与 对结节细胞的放电和动作电位时程至关重要。我们建议直接检验这一假设 使用SK1、SK2和SK3通道的零突变模型。我们建议的研究将大大扩展我们的 了解单个钙离子通道和SK通道的具体功能。事实上，对世界的新见解 心房特异性和起搏组织特异性离子通道可能提供靶向这些通道的新手段 而不会干扰心脏组织的兴奋性。

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.