Dynamic Calmodulin Regulation of Na Channels

描述（申请人提供）：钙调蛋白（Calmodulin, CaM）对四畴通道的调控最早出现在CaV通道中，并已被证明具有丰富的机制和生物学意义。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。考虑到CaV和NaV通道序列的相似性，人们产生了有趣的怀疑，即NaV通道也可能表现出这种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Ca2+和/或CaM对NaV通道的功能影响相当微妙，主要局限于稳态失活（h%）曲线的几mv移动。然而，在Ca2+和/或CaM调节的推定结构决定因素上的通道性NaV突变确实赋予了严重的疾病表型。一个潜在的罪魁祸首是，与CaV通道不同，NaV通道不传导Ca2+，因此它们不能直接触发Ca2+反应。相反，NaV场使用全细胞透析在几分钟或更长时间内强直地操纵Ca2+水平。该领域是否具有对Ca2+和/或CaM的脱敏反应的特征，与短期和较大的生理效应具有较差的相似性？在这里，我们使用一种不同的方法来动态扰动Ca2+，我们的初步数据揭示了在该领域长期寻求的东西：快速和强大的Ca2+/ cam依赖性NaV通道失活（CDI）。现在允许的进步可能会彻底改变CaM对NaV通道调节的理解。这个项目将通过三个具体目标开启一个激动人心的发现时代。(1)揭示Ca2+/ cam介导的NaV通道家族快速调控的存在。(2)阐明cam介导的NaV通道动态调控机制。有了长期寻求的NaV监管的稳健读数，Aim 2将以独特的姿态剖析监管的机制基础。(3)评估cam介导的Nav通道调控的更广泛的生物学影响。上述机制的进展包含了Aim 3将探讨的许多生物学意义。总而言之，该项目将促进Ca2+对NaV通道的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Ca2+调节与某些通道病变联系起来，并可能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.