Project Summary/Abstract Voltage-gated sodium (Nav) channels are integral membrane proteins that selectively conduct Na+ ions across cell membranes. They are associated with cardiovascular, neurological, and psychiatric disorders and are the molecular targets of widely used antiarrhythmic, anticonvulsant drugs. The human Nav1.5 channel generates cardiac action potentials and is associated with life-threatening arrhythmias. The atomic structure of Navs was first obtained from a prokaryotic NavAb channel in 2011, and then more eukaryotic Nav structures were solved by cryo-EM in recent years, including the human cardiac Nav1.5 channel. Both the prokaryotic and eukaryotic Nav channels are very similar in structure, including their selectivity filters, ion permeation pores, voltage sensors, and pharmacological profiles. Most recently, the resting and activating conformations of NavAb channels were obtained by combining the function-dependent cross-linking and cryo-EM, which provided basic molecular frameworks to further investigate the mechanisms of voltage gating and drug modulation. My project aims to reveal dynamic behaviors of the selectivity filter pores and voltage sensors in NavAb and Nav1.5 channels and the effects of permeant/blocking ions, gating voltages, and drug molecules on them. We will implement the cutting-edge single molecule fluorescence resonance energy transfer (smFRET) approach to achieve these proposed studies. Specifically, we will use both the model NavAb and human Nav1.5 channels to (a) uncover the conformational flexibilities and dynamics of the Na selectivity filter and elucidate how it can selectively conduct Na+ over cations such as K+ and Ca2+; (b) define the roles of selectivity filters in slow inactivation, and understand how antiarrhythmic drugs like lidocaine and flecainide alter them to inhibit channel function; (c) reveal the real time conformational transitions and dynamics of the voltage sensor and channel gate in NavAb and Nav1.5 channels that is directly driven by the electrical potential to elucidate the mechanism underlying voltage sensing and gating. We have obtained very exciting preliminary data on the NavAb channel, which strongly justified the significance and feasibility of the proposed studies. In the resubmission, we further made the key technical advances by establishing the unnatural amino acid incorporation method, which allows us to label the human Nav1.5 channel with fluorophores for smFRET studies. With the Nav1.5 channel, we will validate the key findings made on the NavAb channel and reveal the dynamic properties that are unique for eukaryotic Navs. My studies will provide fundamental mechanistic insights into the ion selectivity, voltage gating, and drug modulation of Nav channels, which will have broad implications on other channels and transporters by providing both conceptual advances and novel methodologies.