Calcium modification of voltage gated sodium channels

项目摘要 电压门控离子通道对于位于全身的可兴奋细胞的动作电位是必不可少的 （中枢神经系统、平滑肌、心脏和骨骼肌）。功能丧失、不适当或不合时宜的功能， 都可能导致疾病离子通道基因中的许多个体点突变或 辅助蛋白与疾病有关，其中一些可能危及生命。很多疾病- 相关突变位于或靠近辅助蛋白结合位点。因此，我们付出了巨大的努力， 许多研究者提出了离子通道门控修饰的机制。 已经确定的是，Ca 2+可以改变离子通道功能，并且Ca 2+敏感蛋白钙调素 (CaM)在这些过程中发挥着重要作用。结构研究已经确定了许多不同的钙离子 通道相互作用;然而，假设的生理功能和对这些数据的解释通常是 争议早期的研究依赖于在不存在或存在Ca 2+的情况下测量离子通道功能， 这产生了似乎完全不同的结果。随后的研究揭示了Ca 2 +- 驱动的修饰是复杂的，并且可能涉及多种辅助蛋白。 我以前发现了钙调素和部分电压门控钠离子之间的高亲和力相互作用， 直接负责灭活传导的通道。我利用我深入的结构 在某些实施方案中，本发明提供了一种表征以损害CaM相互作用而不赋予通道功能额外的修饰的方法。 这是一个值得注意的成就，因为通道的这一部分在过程中经历了快速的构象变化。 每个功能周期。正因为如此，我第一次可以清楚地将修饰的钠离子通道 减少钙调素结合的功能。我的数据表明钙调素减少的钠离子通道 相互作用需要更长的时间从失活状态恢复。 考虑到我的结构/功能研究结果与文献提出了一个范例钙调素促进 灭活后回收率（CFRI）。正如我的论文和科学数据所证明的那样，CaM参与了 具有高亲和力的几种钠通道亚型的失活门控，提示了一种独特的 调控我的发现与其他报道直接冲突， (CDI)[Ca 2 +]不敏感。这些相反的模型产生于知识差距，关于（i）动力学速率 的钙调素相互作用和（ii）每一个钙调素相互作用的确切作用，在一个可兴奋的细胞，包含振荡 [Ca2+].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可兴奋细胞中CaM-离子通道相互作用的作用。 重要的是，我们然后利用这些知识来设计定制的小分子（NMR方法的SAR）， 改变辅助蛋白相互作用的动力学，目的是调节通道门控。这项工作将测试 模型的Ca 2+修饰离子通道功能，并探索新的战略，治疗通道病。

PROJECT SUMMARY Voltage-gated ion channels 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 ion channel or accessory proteins have been associated with disease, some of which can be life threatening. Many disease- associated mutations are at or near accessory protein binding sites. Therefore, significant effort has been put forth by many investigators to characterize mechanisms of ion channel gating modification. It is well established that Ca2+ can alter ion channel function, and the Ca2+ sensing protein calmodulin (CaM) has a prominent role in these processes. Structural investigations have identified many distinct CaM-ion channel interactions; however, the posited physiological function and interpretation of this data is often controversial. Early studies relied on measuring ion channel function in the absence or presence of Ca2+ and this has generated seemingly disparate results. Subsequent investigation revealed the mechanism(s) of Ca2+- driven modification are complex and can involve multiple accessory proteins. I previously identified a high-affinity interaction between CaM and part of a voltage-gated sodium channel that is directly responsible for inactivating conduction. I leveraged my in-depth structural characterization to impair the CaM interaction without conferring additional modification to channel 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 sodium channel function to reduced CaM binding. My data demonstrate that sodium channels with this reduced CaM interaction require longer to recover from the inactivated state. Considering my structure/function findings with literature suggests a paradigm of CaM Facilitated Recovery from Inactivation (CFRI). As demonstrated in my papers and scientific data, CaM engages the inactivation gate of several sodium channel isoforms with high affinity, suggesting a unique 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-ion channel interactions in excitable cells. Importantly, we then leverage this knowledge to design custom small molecules (SAR by NMR approach) that alter the kinetics of accessory protein interactions, with a goal of tuning channel gating. This work will test models of Ca2+ modification of ion channel function, and explore novel strategies for treating channelopathies.