Dynamic Calmodulin Regulation of Na Channels

描述（由申请人提供）：钙调素（CaM）对四域通道的调节首先出现在CaV通道中，并且已证明在机械和生物学上都是丰富的。我们的实验室是揭开CaV通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，引起了有趣的怀疑，NaV通道也可能表现出这样的CaM调节。这种可能性是最有吸引力的，因为NaV通道具有广泛的生物学和临床影响，包括精神状态（癫痫）、肌肉收缩（肌强直和心律失常）和感觉（神经性疼痛）。生物化学和结构生物学都强调了这种相似性，但报道的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mV位移。然而，在Ca 2+和/或CaM调节的推定结构决定因素处的通道病NaV突变确实赋予严重的疾病表型。明显断开的一个潜在原因是，与CaV通道不同，NaV通道不传导Ca 2+，因此它们不能直接触发Ca 2+反应。相反，NaV领域已经使用全细胞透析来在几分钟或更长时间内对Ca 2+水平进行紧张性操纵。该领域是否可以表征对Ca 2+和/或CaM的脱敏反应，与短期和更大的生理效应相似性较差？在这里，我们使用一种不同的方法来动态扰动Ca 2+，我们的初步数据揭示了该领域长期寻求的东西> NaV通道（CDI）的快速和稳健的Ca 2 +/CaM依赖性失活。现在允许的进展可能会彻底改变对NaV通道的CaM调节的理解。该项目将通过三个具体目标开创一个令人兴奋的发现时代。(1)揭示NaV通道家族中存在快速Ca 2 +/CaM介导的调节。(2)阐明CaM介导的NaV通道动态调节机制。随着长期寻求的对NaV监管的有力解读，Aim 2将独特地剖析监管的机械基础。(3)评估CaM介导的Nav通道调节的更广泛生物学影响。上述机制上的进展具有许多生物学意义，目标3将探索。总而言之，该项目将促进NaV通道的Ca 2+调节取得前所未有的进展。同样，这一提议将明确地将NaV通道的Ca 2+调节与某些通道病联系起来，也许与相关但更普遍的疾病形式联系起来。

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.