Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K channels. Particularly, in the prokaryotic KcsA channel the K conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation p of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.