PROJECT SUMMARY/ABSTRACT Ion channels in the voltage-gated ion channel (VGIC) superfamily play pivotal roles in virtually all physiological processes. This proposal delves into essential constituents of this superfamily, focusing on elucidating the mechanisms governing voltage sensing and electromechanical coupling of these proteins in pathophysiological contexts. Our investigation focuses on discerning the regulatory interplay between the voltage-sensing domain (VSD) of VGICs and the lipid-ordered membrane domain (OMD) enriched with cholesterol. Recent findings from our laboratory have unraveled the significant influence of OMD on modulating membrane excitability in somatosensory dorsal root ganglion (DRG) neurons. A reduction in OMD dimensions, leading to augmented native ionic currents of pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, emerges as a contributing factor in neuropathic pain and inflammatory pain. These distinct lipid nanodomains exercise direct control over the voltage sensor of HCN channels, consequently influencing channel opening. This aspect of HCN channel regulation, integral to neuronal excitability and cardiac pacemaking, necessitates in-depth investigation. Our biophysical approach employs patch-clamp fluorometry (PCF) combined with fluorescence lifetime imaging microscopy (FLIM) to measure voltage-sensor conformational dynamics within native lipid environments. Leveraging genetic-code expansion featuring noncanonical amino acids and bio- orthogonal fluorescence labeling through click chemistry enables site-specific labeling and facilitates Förster resonance energy transfer (FRET) measurements. Furthermore, our approach includes an improved transition metal FRET (tmFRET) technique in conjunction with phasor plot FLIM, with the primary goal of investigating the potential intermediate states of VGIC voltage sensors. By successfully implementing this approach, we intend to discern the influence of OMD localization, lipid composition, and disease-associated mutations on these intermediate voltage sensor states. This extensive investigation holds the promise of significantly deepening our understanding of the voltage-sensing mechanism of VGIC in physiological processes.