Calcium modification of voltage gated sodium channels

项目摘要 电压门控钠通道（NaVs）是位于全身的可兴奋细胞动作电位的必需通道。 身体（中枢神经系统、平滑肌、心脏和骨骼肌）。损失，不适当的或不合时宜的 功能，可以导致或有助于疾病。NaV或NaV基因中的许多个体点突变 辅助蛋白与疾病有关;其中一些可能危及生命。许多疾病 相关突变位于NaV辅助蛋白结合位点处或附近;因此， 许多研究人员提出了离子通道门控的基本机制 在生理学和疾病中。 Ca2+改变NaV的功能已被证实，Ca2+敏感蛋白钙调素（CaM）具有 在这一过程中发挥了重要作用。结构研究已经确定了几种不同的CaM-NaV相互作用。 然而，假设的生理功能和数据的解释是有争议的。早期的研究依赖于 在不存在或存在Ca2+的情况下测量NaV功能，并产生看似不同的结果。 随后的研究揭示了Ca2+驱动的NaV修饰的机制是复杂的，涉及 多种辅助蛋白，从而使许多数据模糊不清。 最近，我发现了CaM和部分NaV之间的高亲和力相互作用， 用于使NaV传导失效。我能够利用我深入的结构特征来削弱 这是一种相互作用，而不赋予NaV功能额外的修改。这是一个令人瞩目的成就， 通道的这一部分在每个功能循环期间经历快速的构象变化。所以我 可以第一次清楚地将修改的NaV功能归因于减少的CaM结合。我的数据显示 具有这种减少的CaM相互作用的通道需要更长的时间才能从失活状态恢复。 考虑到我的结构/功能的研究结果与现有的文献提出了一个范例钙调素 促进灭活恢复（CFRI）。正如我最近的论文和初步数据所证明的那样，CaM 以高亲和力接合几种NaV同种型，表明了一种通用的调节模型。我的发现在 直接冲突与其他报告，crossing模型的钙调素依赖性失活（CDI）和[Ca 2 +]不敏感性。 这些相反的模型产生于知识差距，关于（i）钙调素相互作用的动力学速率和（ii） 在含有振荡[Ca2 +]的可兴奋细胞中，每个CaM相互作用的确切作用。 我的建议通过独特地结合结构生物学，停流， 动力学和电生理学来剖析CaM-NaV相互作用在可兴奋细胞中的作用。然后我将 探索我是否可以通过设计一个小分子探针来改变特定相互作用的动力学。这项工作将 测试CFRI（生理学和疾病），以及探索治疗NaV通道病的新策略。

PROJECT SUMMARY Voltage-gated sodium channels (NaVs) are essential for action potentials in excitable cells located throughout the body (central nervous system, smooth muscle, heart and skeletal muscle). Loss of, improper, or untimely function, can each cause or contribute to disease. Many individual point mutations in the genes of NaV or accessory proteins have been associated with disease; some of which can be life threatening. Many disease associated mutations are located at or are near NaV accessory protein binding sites; therefore significant effort has been put forth by many investigators to characterize the mechanisms that underlie ion channel gating modification and in physiology and disease. It is well established that Ca2+ alters NaV function, and the Ca2+ sensing protein Calmodulin (CaM) has a prominent role in this process. Structural investigations have identified several distinct CaM-NaV interactions. However, the posited physiological function and interpretation of data are controversial. Early studies relied on measuring NaV function in the absence or presence of Ca2+ and generated seemingly disparate results. Subsequent investigation revealed the mechanism(s) of Ca2+driven NaV modification are complex and involve multiple accessory proteins, thereby rendering much of the data ambiguous. Recently, I identified a high-affinity interaction between CaM and part of NaV that is directly responsible for inactivating NaV conduction. I was able to utilize my in-depth structural characterization to impair the interaction without conferring additional modification to NaV function. This is a notable accomplishment given this part of the channel undergoes rapid conformational change during each functional cycle. Because of this, I could for the first time clearly attribute modified NaV function to reduced CaM binding. My data demonstrate that channels with this reduced CaM interaction require longer to recover from the inactivated state. Considering my structure / function findings with available literature suggest a paradigm of CaM Facilitated Recovery from Inactivation (CFRI). As demonstrated in my recent papers and preliminary data, CaM engages several NaV isoforms with high affinity, suggesting a universal model of regulation. My findings are in direct conflict with other reports that posit models of CaM Dependent Inactivation (CDI) and [Ca2+] insensitivity. These opposing models arise from knowledge gaps regarding (i) the kinetic rates of CaM interactions and (ii) the precise role of each CaM interaction in an excitable cell that contains oscillating [Ca2+]. My proposal addresses these knowledge gaps by uniquely combining structural biology, stopped-flow kinetics, and electrophysiology to dissect the roles of the CaM-NaV interactions in excitable cells. I will then explore if I can alter the kinetics of specific interactions by engineering a small molecule probe. This work will test CFRI (physiology and disease), as well as explore novel strategies for treating NaV channelopathies.