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

Abstract

ABSTRACTVoltage sensors are crucial for the electromechanical coupling of rapid delayed rectifier hERG channels in cardiac pacemaking. They detect and respond to changes in membrane voltage by traversing across the membrane electric field. Mutations in voltage-sensing arginines of hERG associated with Long QT syndrome induce distinct channel gating behaviors, yet knowledge about voltage sensing in these mutants remains limited. The goal of this research is to elucidate potential intermediate states and conformational heterogeneity of voltage sensors, by leveraging patch-clamp electrophysiology, fluorescence lifetime imaging microscopy (FLIM), and transition metal Förster resonance energy transfer (tmFRET). Facilitated by dual stop-codon mediated noncanonical amino acid incorporation, along with click chemistry for site-specific labeling, we preformed FLIM-tmFRET measurements on the hERG voltage sensor. Analysis of lifetime changes of the FRET donor L-acridonyl-alanine (Acd) using the phasor approach of FLIM revealed multiple FRET states in mutant hERG channels, in contrast to the single high FRET state observed in wild-type hERG channels. The presence of these intermediate FRET states correlates with the specific location of mutations, suggesting distinct voltage-sensor conformations. This study provides novel insights into cardiac channelopathies and the underlying structural mechanisms of voltage sensing in cardiac arrhythmias.