The human ether-à-go-go encoded potassium channel (Kv11.1) is essential for cardiac repolarization, and neutralization of its S4 voltage-sensing arginines is a known cause of congenital long-QT syndrome (LQTS). Although such mutations are known to impair gating, the structural basis of altered voltage sensing remains unclear. We combined live-cell fluorescence lifetime imaging microscopy with transition metal FRET (FLIM-tmFRET), site-specific noncanonical amino-acid labeling, and molecular dynamics (MD) simulations to investigate how two mutations, R531Q and R528Q, reshape S4 dynamics. FLIM-tmFRET revealed that wild-type channels predominantly occupy a high-FRET state, consistent with an activated voltage sensor, whereas both mutants stabilized additional intermediate FRET states. Electrophysiology showed that R531Q causes a strong depolarizing shift and biphasic activation, while R528Q primarily alters activation kinetics with a modest shift in the conductance-voltage relationship. MD simulations provided structural context: equilibrium and steered trajectories indicated that mutant S4 helices adopt more elevated and tilted conformations compared to the wild type. R531Q also showed altered local secondary structure that may hinder upward displacement. Free-energy profiles from umbrella sampling confirmed stabilization of intermediate conformations and reduced stability of the fully activated state. Together, these results demonstrate that S4 arginine neutralizations within the charge transfer center shift the conformational balance of the hERG voltage sensor toward metastable intermediates, thereby disrupting gating energetics and kinetics. This integrative approach, combining live-cell FLIM-tmFRET with atomic simulations, defines how pathogenic mutations perturb voltage sensing and highlights the power of linking experimental and computational strategies to dissect ion-channel gating mechanisms.
Quach et al. (Sun,) studied this question.