Calcium regulation and dysregulation of cardiac ion channels

描述(由申请人提供)：本提案的目的是确定细胞内钙离子的变化如何调节心肌膜兴奋性。然而，钙离子调节失调会导致心律失常。考虑到潜在的病理生理后果，许多调控机制已经进化到控制钙离子进入也就不足为奇了。其中包括CaV1.2 L类钙通道的钙依赖失活(CDI)，这是先前提出的重点和我们在CDI研究中新描述的机制。在这里，我们关注钙离子通过影响多个通道的门控来塑造心脏动作电位和影响兴奋性的新方法。提出了三个具体目标。在第一个目标中，我们基于初步数据表明，通过核磁共振波谱确定NaV1.5 C末端(CT)的结构是可行的。我们将确定NaV1.5可能的钙调控区的原子结构，这将使我们能够解决关于NaV1.5是否以及如何受钙调控的争议。这些实验还将提供对原子结构的洞察和解释NaV1.5的关键C末端(CT)和III-IV细胞内连接子的功能。此外，我们将通过这些结构研究阐明特定遗传性致心律失常突变的分子机制。在第二个目的中，我们认识到CaM只是一个调节离子通道功能的钙结合蛋白(CaBP)；在体内，离子通道功能对钙的敏感性可能反映了作用于不同通道决定因素的多个CaBP的汇聚。我们探索了KChIP2对CaV1.2钙通道的新的调节作用，KChIP2是一种CABP，以前被证明可以调节K+电流，但尚未有报道控制钙通道。第三个目标集中在钙离子如何通过激活对钙离子敏感的效应物，如激酶来调节离子通道。在我们先前关于CaMKII与CaV1.2钙通道相互作用以控制钙依赖促进(CDF)的证据的基础上，我们建议探索分子模拟模型的结果，以了解CaMKII和钙通道附属22a亚单位如何相互作用以促进CDF。总之，这些目标的结果将定义新的方法，通过钙离子可以反馈到多个离子通道来调节膜的兴奋性，并表明这些机制的失调是如何导致心律失常的。与公共健康相关：钙信号是心脏电活动的最终共同途径。钙信号的失调参与了心律失常的发生。这项建议阐述了钙离子如何反馈调节心脏离子通道，并检查了离子通道中关键区域的结构，这些区域是遗传性致心律失常突变的基因座。

DESCRIPTION (provided by applicant): The objective of this proposal is to determine how changes in intracellular Ca2+, the ultimate signal of electrical activity, regulate cardiac membrane excitability. Dysregulation of Ca2+, however, contributes to cardiac arrhythmias. Given the potential pathophysiological consequences, it is not surprising that many regulatory mechanisms have evolved to control Ca2+ entry. These include Ca2+-dependent inactivation (CDI) of CaV1.2 L- type Ca2+ channels the focus of the previous proposal and mechanisms newly described by us that derived from our studies on CDI. Here we focus on novel means by through which Ca2+ influences gating of multiple channels to shape the cardiac action potential and influence excitability. Three specific aims are proposed. In the first aim, we build upon preliminary data showing that structural determination of the Nav1.5 C terminus (CT) is feasible by NMR spectroscopy. We will determine the atomic structure of the putative Ca2+ regulatory regions of Nav1.5, which will allow us to address the controversy about whether and how Nav1.5 is regulated by Ca2+. These experiments will also provide insight into atomic structure and explain function of the critical C terminus (CT) and III-IV intracellular linker of Nav1.5. Further, we will illuminate molecular mechanisms of specific inherited arrhythmogenic mutations through these structural studies. In the second aim, we recognize that CaM is but one Ca2+ binding protein (CaBP) that regulates ion channel function; in vivo Ca2+-sensitivity of ion channel function might reflect a convergence of multiple CaBPs acting upon different channel determinants. We explore novel regulation of CaV1.2 Ca2+ channels by KChIP2, a CaBP previously shown to modulate K+ currents, but not yet reported to control Ca2+ channels. The third aim focuses on how Ca2+ regulates ion channels through the activation of Ca2+-sensitive effectors, such as kinases. Building upon our previous demonstration that CaMKII interacts with CaV1.2 Ca2+ channels to control Ca2+- dependent facilitation (CDF), we propose to explore the consequences of a molecular mimicry model to understand how CaMKII and the Ca2+ channel accessory 22a subunit interact to foster CDF. Together, results from these aims will define new means by which Ca2+ can feedback on multiple ion channels to regulate membrane excitability and show how dysregulation of these mechanisms is arrhythmogenic. Public Health Relevance: Ca2+ signaling is the final common pathway of electrical activity in the heart. Dysregulation of Ca2+ signaling contributes to cardiac arrhythmias. This proposal addresses how Ca2+ feeds back to regulate cardiac ion channels and examines the structure of critical domains in ion channels that are loci for inherited arrhythmogenic mutations.