Project Summary/Abstract Voltage-gated sodium (Nav) channels are associated with cardiovascular, neurological, and psychiatric disorders and are the molecular targets of widely used antiarrhythmic, anticonvulsant drugs. Our project aims to study the structural dynamics of the human Nav1.5 channel, which generates action potentials in cardiomyocytes and is associated with life-threatening arrhythmias. High-resolution structures of many voltage- gated sodium channels, including Nav1.5, have revealed the molecular details of selectivity filter pores, voltage sensors, and drug-binding sites. Recently, NavAb structures chemically locked at the resting and activating states were obtained by cryo-EM, providing a molecular framework to understand the structural basis of voltage sensing and gating. Based on these high-resolution structures, my project proposed investigating the dynamic behaviors of Nav channel structures using the cutting-edge single-molecule fluorescence resonance energy transfer (smFRET) technique. We aim to uncover the conformational changes and kinetics of Nav channels at critical sites, including selectivity filters, voltage sensors, and drug binding sites, which are vital to understanding how Nav channels select Na+ over other cations, how membrane voltages alter the conformational landscape of voltage sensors to gate channel pores, and how drug molecules alter Nav structures to modulate their function. SmFRET measurements on Nav channels, proposed as key studies in my project, are currently performed on a customized total internal reflection fluorescence (TIRF) microscope in my lab. This proposal seeks support to acquire the Nano-imager (Oxford Nano-Imaging Inc), an automatic microscope that integrates hardware, control, and analysis software to perform more robust smFRET studies. The autofocus system, the piezo stage, and the temperature controller built into the Nanoimager will allow us to resolve conformational dynamics as fast as 1 millisecond, obtain accurate FRET efficiency measurements for structural modeling, and even examine conformational dynamics of voltage-gated ion channels in living cells. Moreover, the Nano-imager eliminates complex hardware alignments and software settings required by my customized TIRF microscope, so even fresh undergraduate students can be trained to work on it within 2 days. In summary, acquiring the Nanoimager will significantly boost our research productivity in the following 3 project years and broadly impact UMKC students by providing them with hands-on experience with this cutting- edge research equipment.