Dynamic Calmodulin Regulation of Na Channels

Na 通道的动态钙调蛋白调节

• 批准号: 8217079
• 负责人: DAVID T YUE
• 金额: $ 39.89万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8217079
• 关键词: Allosteric Regulation; Arrhythmia; Binding; Biochemistry; Biological; Biology; Calmodulin; Cells; Clinical; Data; Dialysis procedure; Disease; Engineering; Epilepsy; Esthesia; Exhibits; Family; Fluorescence Resonance Energy Transfer; Hand; Health; Investigation; Laboratories; Life; Link; Lobe; Mediating; Molecular; Muscle; Muscle Contraction; Mutation; Myotonia; Physiological; Physiology; Protein Isoforms; Recombinants; Regulation; Reporting; Research; SCN1A protein; Screening procedure; Seizures; Signal Transduction; Site; System; Testing; Translating; Variant; base; disease phenotype; improved; innovation; interest; novel; painful neuropathy; research study; response; stoichiometry; structural biology; trafficking; voltage

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. PUBLIC HEALTH RELEVANCE: Ca2+ and/or calmodulin regulation of voltage-gated Na channels is poised to impact diverse aspects of physiology, as well as diseases like epilepsy/seizure, muscle myotonia/arrhythmia, and neuropathic pain. Yet, the severe disease phenotypes relating to Na channels seem incongruous with the subtle functional effects of Ca2+ and/or calmodulin seen when Na channels are intentionally studied in isolation. This proposal likely overcomes a flaw in the way isolated Na channels have been studied thus far, thereby revealing the full-bodied effects of Ca2+ regulation on Na channels, and unlocking an era of rapid advance in linking Na channel regulation to biology, disease, and potentially therapy.

描述（由申请人提供）：四结构域通道的钙调蛋白（CaM）调节首先出现在CaV通道中，并已在机械和生物学方面被证明是丰富的。我们的实验室是揭开 CaV 通道发现这一令人兴奋的篇章的领导者之一。鉴于 CaV 和 NaV 通道的序列相似性，人们怀疑 NaV 通道也可能表现出这种 CaM 调节。考虑到 NaV 通道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），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，但所报道的 Ca2+ 和/或 CaM 对 NaV 通道的功能影响相当微妙，大多局限于稳态失活 (h%) 曲线的几 mV 变化。然而，Ca2+ 和/或 CaM 调节的假定结构决定因素的通道病 NaV 突变确实会导致严重的疾病表型。造成明显断开的一个潜在原因是，与 CaV 通道不同，NaV 通道不传导 Ca2+，因此它们不能直接触发 Ca2+ 响应。相反，NaV 领域使用全细胞透析来调节 Ca2+ 水平，持续几分钟或更长时间。该领域是否可以表征对 Ca2+ 和/或 CaM 的脱敏反应，与短期和更大的生理效应的相似性较差？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该领域长期寻求的东西> NaV 通道 (CDI) 的快速和稳健的 Ca2+/CaM 依赖性失活。现在允许的进步可能会彻底改变对 NaV 通道的 CaM 监管的理解。该项目将通过三个具体目标迎来一个激动人心的发现时代。 (1) 揭示 NaV 通道家族中存在快速 Ca2+/CaM 介导的调节。 (2)阐明CaM介导的NaV通道动态调节机制。有了长期寻求的 NaV 监管的稳健读数，Aim 2 将独特地准备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。上述机制进步具有许多生物学意义，Aim 3 将探索这些意义。总而言之，该项目将促进 NaV 通道的 Ca2+ 调节取得前所未有的进展。此外，该提案将明确将 NaV 通道的 Ca2+ 调节与某些通道病联系起来，或许还与相关但更普遍的疾病形式联系起来。 公共健康相关性：Ca2+ 和/或钙调蛋白对电压门控 Na 通道的调节可能会影响生理学的各个方面，以及癫痫/癫痫发作、肌肉强直/心律失常和神经性疼痛等疾病。然而，与 Na 通道相关的严重疾病表型似乎与有意单独研究 Na 通道时观察到的 Ca2+ 和/或钙调蛋白的微妙功能效应不一致。这一提议可能克服了迄今为止研究孤立Na通道的方式中的缺陷，从而揭示了Ca2+调节对Na通道的全面影响，并开启了将Na通道调节与生物学、疾病和潜在治疗联系起来的快速发展时代。