Calcium modification of voltage gated sodium channels

项目总结 电压门控钠通道(NAV)是遍及全身的可兴奋细胞的动作电位所必需的。 身体(中枢神经系统、平滑肌、心脏和骨骼肌)。丢失、不当或不合时宜 功能，每一种都可能导致或促成疾病。许多NAV或NAV基因的个体点突变 辅助蛋白与疾病有关；其中一些可能危及生命。多种疾病 相关突变位于NAV辅助蛋白结合部位或其附近；因此需要做出重大努力 已经被许多研究人员用来描述离子通道门控背后的机制 以及在生理和疾病方面的改变。 已经证实，钙离子改变NAV功能，而钙离子敏感蛋白钙调蛋白(CaM)具有 在这一进程中发挥着突出的作用。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 在没有或存在钙离子的情况下测量NAV功能，产生了看似不同的结果。 随后的研究发现，钙离子驱动的NAV修饰的机制(S)是复杂的，涉及 多个辅助蛋白，从而使许多数据模糊不清。 最近，我发现了CaM和NAV的一部分之间的高亲和力相互作用，这是直接负责的 去激活NAV传导。我能够利用我深入的结构特征来削弱 交互，而不会对NAV功能进行额外修改。这是一项值得注意的成就 在每个功能周期中，通道的这一部分经历了快速的构象变化。正因如此，我 可以第一次明确地将修改的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交互的通道需要更长的时间才能从停用状态中恢复。 考虑到我的结构/功能和现有文献的发现，提出了一种CaM的范式 促进从失活中恢复(CFRI)。正如我最近的论文和初步数据所表明的那样，Cam 与几种高亲和力的NAV亚型结合，提出了一种通用的调节模型。我的研究结果发表在 与其他提出钙调素依赖失活(CDI)和[Ca~(2+)]不敏感模型的报道直接冲突。 这些对立的模型源于关于(I)凸轮相互作用的动力学速率和(Ii) 每一个CaM相互作用在含有振荡[Ca~(2+)]的可兴奋细胞中的确切作用。 我的建议通过独特地将结构生物学、停滞流 动力学和电生理学，剖析CaM-NAV相互作用在可兴奋细胞中的作用。那我会的 探索我是否可以通过设计一个小分子探测器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.