Dynamic Calmodulin Regulation of Na Channels

说明(申请人提供)：钙调蛋白(CaM)对四结构域通道的调节最先出现在Cav通道中，并被证明在机制和生物学上都很丰富。我们的实验室一直是揭开CAV频道发现这一激动人心篇章的领导者之一。鉴于CAV和NAV通道的序列相似性，出现了耐人寻味的怀疑，NAV通道可能也表现出这种CaM调节。考虑到NAV通道的广泛生物学和临床影响，包括镇静(癫痫)、肌肉收缩(肌强直和心律失常)和感觉(神经病理性疼痛)，这种可能性最具吸引力。生物化学和结构生物学都强调了这种相似性，但已报道的Ca~(2+)和/或CaM对NAV通道的功能影响相当微妙，主要限于稳态失活(h%)曲线的几个mV位移。然而，在钙离子和/或CaM调节的假定结构决定因素上的通道病态NAV突变确实赋予了严重的疾病表型。造成这种明显脱节的一个潜在原因是，与Cav通道不同，NAV通道不传导钙离子，因此它们不能直接触发钙离子反应。取而代之的是，NAV领域使用全细胞透析来调节钙离子水平，持续几分钟或更长时间。磁场是否表现出对钙离子和/或钙调素的不敏感反应，与短期和更大的生理效应没有什么相似之处？在这里，我们使用一种不同的方法来动态干扰钙离子，我们的初步数据揭示了该领域长期寻求的一种现象&快速而强大的依赖钙/钙调素的NAV通道失活(CDI)。现在允许的进展可能会彻底改变人们对钙调素调节NAV通道的理解。该项目将通过三个具体目标开启一个令人兴奋的发现时代。(1)揭示钙/钙调素介导的跨NAV通道家族快速调节的存在。(2)阐明钙调素对NAV通道的动态调节机制。随着长期寻求的资产净值监管读数的强劲出炉，Aim 2将独一无二地准备好剖析监管的机制基础。(3)评估CaM介导的NAV通道调节的更广泛的生物学影响。上述机制上的进展具有许多生物学意义，目标3将对此进行探索。总而言之，该项目将促进NAV通道的钙离子调节取得前所未有的进展。此外，这一建议将明确地将NAV通道的钙调节与某些通道病联系起来，或许还与相关的、但更普遍的疾病有关。

DESCRIPTION (provided by applicant): Calmodulin (CaM) regulation of four-domain channels first emerged in CaV channels, and has proved rich both mechanistically and biologically. Our laboratory has been one of the leaders in unveiling this exciting chapter of CaV channel discovery. Given the sequence similarity of CaV and NaV channels, intriguing suspicions arose that NaV channels might also exhibit such CaM regulation. This possibility was most attractive, given the wide-ranging biological and clinical impact of NaV channels, which include mentation (epilepsy), muscle contraction (myotonia and arrhythmias), and sensation (neuropathic pain). Biochemistry and structural biology both underscored this similarity, but the reported functional effects of Ca2+ and/or CaM on NaV channels have been rather subtle, mostly limited to several-mV shifts of steady-state inactivation (h%) curves. Yet, channelopathic NaV mutations at putative structural determinants of Ca2+ and/or CaM regulation do confer severe disease phenotypes. One potential culprit for the apparent disconnect is that, unlike CaV channels, NaV channels do not conduct Ca2+, so they cannot directly trigger Ca2+ responses. Instead, the NaV field has used whole-cell dialysis to tonically manipulate Ca2+ levels, over several minutes or longer. Could the field be characterizing desensitized responses to Ca2+ and/or CaM, with poor similarity to short-term and larger physiological effects? Here, we use a different approach to dynamically perturb Ca2+, and our preliminary data unveil something long sought in the field>rapid and robust Ca2+/CaM-dependent inactivation of NaV channels (CDI). The advances now permitted may revolutionize understanding of the CaM regulation of NaV channels. This project will usher in an exciting era of discovery via three specific aims. (1) To unveil the existence of rapid Ca2+/CaM-mediated regulation across the family of NaV channels. (2) To elucidate the mechanism of dynamic CaM-mediated regulation of NaV channels. With long-sought robust readouts of NaV regulation in hand, Aim 2 will be uniquely poised to dissect the mechanistic underpinnings of regulation. (3) To assess the broader biological impact of CaM-mediated regulation of Nav channels. The mechanistic advances above hold numerous biological implications that Aim 3 will explore. In all, this project will facilitate a period of unprecedented progress in the Ca2+ regulation of NaV channels. As well, this proposal will explicitly link Ca2+ regulation of NaV channels to certain channelopathies, and perhaps to related but more generalized forms of disease.