Voltage Sensor Conformations Induced by LQTS-associated Mutations in hERG Potassium Channels

ABSTRACT Voltage sensors are essential for electromechanical coupling in hERG K + channels, critical to cardiac rhythm. These sensors respond to membrane potential changes by moving within the transmembrane electric field. Mutations in hERG voltage-sensing arginines, associated with Long-QT syndrome, alter channel gating, though underlying mechanisms remain unclear. Using live-cell fluorescence lifetime imaging microscopy (FLIM), transition metal FRET (tmFRET), an improved dual stop-codon-mediated strategy for noncanonical amino-acid incorporation, and molecular dynamics (MD) simulations, we identified intermediate voltage-sensor conformations induced by neutralizing key arginines in the charge transfer center. Phasor plot analysis of FLIM data revealed multiple voltage-dependent FRET states in these mutants, in contrast to the single high-FRET state observed in controls. These intermediate FRET states reflect distinct conformations of the voltage sensor, corresponding to predicted structures of voltage sensors in MD simulations. This study provides novel insights into cardiac channelopathies, highlighting a structural mechanism that impairs voltage sensing in cardiac arrhythmias.