Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-55 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by similar to-50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by similar to 1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the a-helical content of the 54-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.

hERG channel gating is associated with relatively slow voltage sensor movement that limits the rate of channel opening and closing. The mechanistic basis underlying the constraints upon sensor movement in these channels is unclear. Here, we have used voltage clamp fluorimetry (VCF) to study the effects of mutations within the S4-S5 linker on voltage sensor movement and its coupling to the pore. Mutations at G546 had two separable effects on activation and deactivation gating. Substitution of G546 with residues possessing different physico-chemical properties all (with the exception of G546C) shifted activation gating by ∼30mV in the hyperpolarizing direction. With the activation shift taken into account, the time constant of ionic current activation was also accelerated. In addition, a number of G546 mutants affected deactivation gating, although the effects of different mutations varied. In the most dramatic case, the G546V mutation induced biphasic deactivation with a pronounced slow component that was voltage-independent. Deletion of the N-terminus accelerated the fast component, but the slow component remained pronounced, suggesting that the slow component was not mediated by altered interaction with the N-terminus. VCF measurements of voltage sensor movement in G546V channels revealed fast and slow components of fluorescence change associated with deactivation, suggesting that the slow component of ionic current deactivation is due to slow voltage sensor return that is uncoupled from charge movement. Taken together, these data suggest: 1) reduced flexibility of the S4-S5 linker helix reduces constraints on voltage sensor movement during activation gating; 2) normal hERG channel closing involves at least two reconfigurations of the voltage sensor that are rate-limiting for pore closure.