Phasor FLIM-FRET Analysis of Voltage Sensor Conformational Heterogeneity in hERG Potassium Channels

Voltage sensors are essential for electromechanical coupling in hERG K+ channels, critical to cardiac rhythm. These sensors detect changes in membrane voltage and move in response to the transmembrane electric field. Mutations in voltage-sensing arginines of hERG, associated with Long QT syndrome, alter channel gating, though mechanisms in these mutants remain unclear. Using fluorescence lifetime imaging microscopy (FLIM), transition metal FRET (tmFRET), dual stop-codon mediated noncanonical amino acid incorporation, and molecular dynamics (MD) simulations, we identified distinct intermediate voltage-sensor conformations caused by these mutations. Phasor plot analysis of the FLIM-tmFRET donor revealed multiple FRET states in mutant hERG channels, in contrast to the single high-FRET state observed in unmutated controls. These intermediate FRET states correspond to specific mutation sites and align with distinct intermediate voltage-sensor conformations identified in MD simulations. This study provides novel insights into cardiac channelopathies, highlighting structural underpinnings underlying voltage sensing in cardiac arrhythmias.