Photochemical determination of sodium channel voltage-dependent gating and composition

电压门控钠通道（NaVs）通过以下途径维持神经元和肌肉组织的电节律： 选择性地控制其同名离子的快速向内通道。基本的NaV复合物是 由260-kDa的成孔α亚基（由NaV1.1-1.9编码）组成，该亚基与β 亚基（A1-A4）。由遗传性突变或通道引起的钠通道功能缺陷 失调是人类疾病的既定原因，并且与婴儿猝死有关， 心律不齐和疼痛综合征。虽然迫切需要更好地了解分子 作为扰动NaV函数的基础，很少有研究工具可用于获得这些见解，这些 持续的技术障碍减缓了许多类型的膜蛋白研究的发现速度。 NaVs已经开始受益于相关细菌NaVs的原子分辨率结构，但除了它们的 从真核细胞通道进化的差异，这些蛋白质在膜的情况下进行分析， 电压，因此检查的构象的相关性是不清楚的。对于真核NaV，关键是 未解决的问题包括静息和失活通道状态之间的结构差异， 失活机制和C-末端的作用，遗传突变对分子门控的影响 事件，以及α-亚基调控NaV通道的分子基础。我们开发了一系列 补充化学生物学研究工具，将提供有关功能的重要新信息 （a）我们已经简化了具有新型点击的遗传编码的交联氨基酸的使用， 化学功能，当与质谱法结合使用时，能够发现 活细胞中的瞬时肽间和肽内相互作用网络。(b)我们开发了一种强大的新 通过这种方法，Cy 3和Cy 5作为非天然氨基酸被编码到活细胞中的膜蛋白中。 这种方法将允许编码的单分子荧光共振能量转移（smFRET）和 直接测量电沉默的构象动力学的膜蛋白在活细胞下， 电压控制(c)真核NaV的全原子计算模型将指导和支持我们的研究。 努力确定NaVs中的构象运动和非共价相互作用。我们建议：（1） 推进钠通道门控的机制理解，重点是失活， 在人类疾病中的巨大作用，以及静息态之间的构象差异和能量耦合 和失活构象;（2）获得光学生成的关键门控状态的相对距离图， 单个NaV以及这些距离如何受到致病突变的影响;以及（3）提供以下基础： α-亚基调控，包括相互作用位点和疾病机制的分子鉴定 导致突变。这三个目标是面向高影响力的发现，并高度相关， 了解多种病理和电信号的分子机制。

Voltage-gated sodium channels (NaVs) maintain the electrical cadence of neurons and muscle tissues by selectively controlling the rapid inward passage of their namesake ion. The essential NaV complex is comprised of a 260-kDa pore-forming alpha subunit (encoded by NaV1.1-1.9) that is partnered with beta subunits (1–4). Defects in sodium channel function resulting from inherited mutations or channel dysmodulation are established causes of human disease, and are associated with sudden infant death, arrhythmia and pain-causing syndromes. While there is an urgent need to better understand the molecular basis for perturbed NaV function, there are few research tools available to obtain these insights, and these persistent technical barriers slow the pace of discovery in the study of many types of and membrane proteins. NaVs have begun to benefit from atomic-resolution structures of related bacterial NaVs, but in addition to their evolved differences from eukaryotic channels, these proteins are analyzed in the absence of membrane voltage and thus the relevance of the conformations examined is unclear. For eukaryotic NaVs, key unaddressed issues include structural differences between the resting and inactivated channel states, the mechanism of inactivation and the role of the C-terminus, the impact of inherited mutations on molecular gating events, and the molecular basis of the NaV channel regulation by -subunits. We have developed a number of complementary chemical biology research tools that will provide essential new information about the function of NaVs: (a) We have streamlined the used of genetically encoded cross-linking amino acids with novel click- chemistry functionality that, when used in combination with mass spectrometry, enables the discovery of transient inter- and intra-peptide interaction networks in live cells. (b) We have developed a powerful new approach whereby Cy3 and Cy5 are encoded as unnatural amino acids into membrane proteins in live cells. This approach will allow for encoded single molecule fluorescence resonance energy transfer (smFRET) and the direct measurement of electrically silent conformational dynamics of membrane proteins in a live cell under voltage control. (c) An all-atom computational model of the eukaryotic NaV that will guide and support our efforts to determine conformational movement and non-covalent interactions in NaVs. We propose to: (1) advance the mechanistic understanding of sodium channel gating, with a focus on inactivation given its outsized role in human disease, and the conformational differences and energetic coupling between resting and inactivated conformations; (2) obtain an optically generated relative distance map of key gating states of single NaVs and how these distances are effected by disease causing mutations; and (3) provide the basis of -subunit regulation, including the molecular identification of interaction sites and mechanisms of disease causing mutations. These three aims are geared towards high impact discovery and are highly relevant to understanding multiple pathologies and the molecular mechanisms of electrical signaling.