PROJECT SUMMARY/ABSTRACT The CNBD family of channels, encompassing hyperpolarization-activated cyclic nucleotide-gated (HCN) and depolarization-activated KCNH members, plays a pivotal role in shaping the electrical properties of the heart. The modulation of HCN channels presents a promising therapeutic avenue for the management of atrial and ventricular arrhythmias by influencing the activity of the sinoatrial node pacemaker. In addition, the human KCNH2 potassium channel (hERG) gene is linked to long QT syndrome (LQTS), an inherited cardiac condition that substantially escalates the risk of ventricular arrhythmias and sudden cardiac death. HCN and KCNH channels share structural features, most notably their voltage-sensing domains (VSD). However, they display divergent channel gating behaviors. Up to this point, a comprehensive mechanism that adequately explains the diversity in their voltage-dependent gating has remained elusive. In particular, the VSD of HCN channels undergoes distinct conformational changes during activation, whose dynamics, nature of intermediate conformational states, and couplings to other domains remain largely uncharacterized. Moreover, like mutations affecting the VSD of HCN channels, we recently uncovered that several LQTS-linked mutations the S4 helix voltage sensor of hERG channels lead to distinctively altered voltage-dependent activation profiles. The underlying mechanism of these disease-causing gating alterations remains unexplored, yet it is central to our understanding of the mechanism of cardiac diseases related to CNBD channels. The overarching objective of this application is to determine the VSD dynamics of recombinant CNBD channels expressed in human cell lines, using an array of concerted biophysical techniques. We will improve the patch-clamp fluorometry (PCF) method to study the structure-function correlation of cardiac voltage-gated channels, paving the way to new treatments of myocardial diseases. Our primary goals also include studying how pivotal regulatory domains modulate channel gating by altering VSD conformations. To quantitatively assess voltage-dependent structural rearrangements, we will leverage transition metal ion Förster resonance energy transfer (tmFRET), noncanonical amino acid integration, and click chemistry for site-specific fluorescence labeling. This strategy will enable us to precisely translate FRET efficiencies into interatomic distances. In parallel, phasor-plot fluorescence lifetime imaging (FLIM) in conjunction with molecular dynamics (MD) simulations will be used to quantify the underlying structural dynamics. Collectively, the proposed studies will delineate the energetics, allostery, and structural foundations of cardiac pacemaking ion channels.