Calcium modification of voltage gated sodium channels

项目总结 电压门控离子通道是遍及全身的可兴奋细胞的动作电位所必需的 (中枢神经系统、平滑肌、心脏和骨骼肌)。功能丧失、不当或不合时宜， 每一种都可能导致或促成疾病。离子通道基因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- 相关突变位于或接近辅助蛋白结合位点。因此，已经作出了重大努力。 第四，许多研究人员对离子通道门控修饰的机制进行了表征。 已有研究表明，钙离子可以改变离子通道功能，而钙离子感应蛋白钙调蛋白 (CAM)在这些过程中发挥着突出的作用。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，假定的生理功能和对该数据的解释通常 有争议的。早期的研究依赖于在没有或存在钙和钙离子的情况下测量离子通道功能 这产生了看似截然不同的结果。随后的研究揭示了钙离子的作用机制(S) 驱动修饰是复杂的，可能涉及多个辅助蛋白。 我之前发现了CaM和部分电压门控钠之间的高亲和力相互作用 直接负责停用传导的通道。我利用我的深度结构 在不对通道功能进行额外修改的情况下削弱凸轮相互作用的特性。 这是一个值得注意的成就，因为这一部分的通道在 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通道 减少凸轮捆绑的功能。我的数据表明，钠通道与这个减少的CaM 交互需要更长时间才能从停用状态恢复。 考虑到我对文献的结构/功能的发现，我提出了一种由CaM促进的范式 从失活中恢复(CFRI)。正如我的论文和科学数据所证明的那样，卡姆从事 几种高亲和力的钠通道亚型的失活门，提示了一种独特的模型 监管。我的发现与其他假设CaM依赖失活模型的报告直接冲突 (CDI)和[钙]不敏感。这些对立的模型产生于关于(I)动力学速率的知识空白 以及(Ii)每个CaM相互作用在包含振荡的可兴奋细胞中的确切作用 [CA2]。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，剖析钙离子通道相互作用在可兴奋细胞中的作用。 重要的是，我们然后利用这一知识来设计定制的小分子(通过核磁共振方法的SAR)， 改变辅助蛋白相互作用的动力学，目的是调节通道门控。这项工作将考验 离子通道功能的钙修饰模型，并探索治疗通道病的新策略。

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.