Functional roles of atrial-specific ion channels in the heart

描述（由申请人提供）： 项目概要/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，并且与栓塞和中风的显著风险相关。事实证明，治疗战略在很大程度上是不够的，这一事实使问题进一步恶化。在上一个资助期间，我们发现了令人惊讶的，但有见地的发现，具有广泛的治疗影响。Cav1.3（a1 D）L型钙通道（LTCC）主要在神经元和神经内分泌细胞中表达，而Cav1.3（a1 D）L型钙通道在心房肌细胞中有显著表达。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+通道在心房中的重要性得到以下揭示的支持：该通道的无效缺失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）结功能障碍。我们进一步证明了Cav1.2和Cav1.3通道与心脏中的小电导Ca 2+激活的K+通道（SK通道）形成多聚体蛋白复合物，这是我们实验室最近首次发现的。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。我们进一步证明了SK2通道通过心肌细胞中的物理桥α-辅肌动蛋白2与Cav1.3和Cav1.2相关联。此外，我们还获得了新的初步数据，表明细胞骨架蛋白在SK2通道的正确膜定位中至关重要。鉴于这些相关数据，我们将直接研究SK通道运输的分子决定因素。此外，新描述的SK通道在起搏组织中的功能作用将直接划定。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，证明了心房细胞Cav通道和Ca 2+激活的K+通道（KCa）之间的功能性时空串扰。嵌入在这些研究结果是相关的范式转变，可以利用开发心房特异性药物治疗房性心律失常。因此，该提案的总体目标是部署新的分子和功能策略，其中许多来自通道机制研究，用于发现心脏中Ca 2+和K+通道的基本和新的可访问领域。我们将使用体外相互作用测定、共聚焦显微镜成像、电子显微镜分析、基因沉默、生物化学研究和功能分析的组合来直接测试该假设。最后，我们假设所有3种亚型的SK通道在SA和AV结细胞中表达，并对结细胞的放电和动作电位持续时间做出重要贡献。我们建议使用SK 1，SK 2和SK 3通道的零突变模型直接检验假设。我们提出的研究将大大扩展我们对单个Ca 2+和SK通道特定功能的理解。事实上，心房特异性和起搏组织特异性离子通道的新见解可能提供新的手段来靶向这些通道，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.