Mutations within the S4-S5 linker alter voltage sensor constraints in hERG K+ channels

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-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼−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 ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-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.