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...