Functional roles of atrial-specific ion channels in the heart

描述(由申请人提供)： 项目摘要/摘要：房颤(房颤)是影响退伍军人人群的最常见的房性心律失常，并与血栓和中风的重大风险有关。事实证明，治疗战略在很大程度上是不够的，这一事实进一步加剧了这一问题。在上一个资助期间，我们发现了令人惊讶但有洞察力的发现，具有广泛的治疗分支。与以往报道的Cav1.3(A1D)L型钙通道主要表达于神经元和神经内分泌细胞不同，我们发现Cav1.3(A1D)型钙通道在心房肌细胞中有显著表达。事实上，与心室肌细胞相比，Cav1.3钙通道在心房肌细胞中优先表达。此外，Cav1.3钙通道在心房中的重要性也得到了证实，该通道的缺失会导致心房兴奋性的显著改变、房性心律失常以及严重的窦房(SA)和房室(AV)结节功能障碍。我们进一步证明，在心脏中，Cav1.2和Cav1.3通道与小电导的钙激活K+通道(SK通道)形成多聚体蛋白复合体，这是我们实验室最近首次发现的。主要耐人寻味和功能重要性的发现是，SK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，SK2通道与Cav1.3和Cav1.2通过心肌细胞中的a-Actinin2这一物理桥梁联系在一起。此外，我们还获得了新的初步数据，这表明细胞骨架蛋白在SK2通道正确的膜定位中起着至关重要的作用。鉴于这些相关数据，我们将直接检验SK渠道贩运的分子决定因素。此外，新描述的SK通道在起搏组织中的功能作用将被直接描述。我们的发现代表了一个统一的分子和细胞机制的开始，该机制显示了心房细胞中Cav通道和钙激活的K+通道(Kca)之间的功能时空串扰。在这些发现中嵌入了相关的范式转变，可以利用这些转变开发针对房性心律失常的治疗房性心律失常的特定药物。因此，该提案的总体主旨是部署新的分子和功能策略，其中许多策略受到通道机制研究的启发，以发现心脏中钙离子和钾离子通道的基本和新的可访问的领域。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的组合来直接验证这一假说。最后，我们假设SK通道的三种亚型均在SA和AV结节细胞中表达，并对结节细胞的放电和动作电位时程起关键作用。我们建议使用SK1、SK2和SK3通道的零突变模型直接检验这一假设。我们提出的研究将极大地扩展我们对单个钙离子和SK通道具体功能的理解。事实上，对心房特异性和起搏组织特异性离子通道的新见解可能为靶向这些通道提供新的手段，而不干扰心室组织的兴奋性。

DESCRIPTION (provided by applicant): 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.