S4-S5 Linker Flexibility Stabilizes hERG Channel Closed States

Abstract

The hERG cardiac K+ channel is characterized by unusually slow activation and deactivation gating kinetics compared to Shaker-like voltage-gated K+ (Kv) channels. In Shaker-like Kv channels movement of the voltage sensor upon depolarization is mechanically transduced by the α-helical S4-S5 linker to S6 activation gate opening. Given the unique gating properties of hERG channels, the details of voltage sensor coupling with the S6 activation gate are of significant interest. We have recently shown that substitution of a glycine residue (G546) within the S4-S5 linker with a leucine residue destabilizes the closed state (left-shifts the V1/2 of activation by ∼50 mV and accelerates the rate of channel opening) and suggested that flexibility of the linker may be a key determinant of the closed-open equilibrium in hERG channels. Here, we have investigated this further by re-introducing glycine residues within the S4-S5 linker (from position 539 to 552) in the G546L background to determine whether flexibility introduced at different positions within the helix restores WT-like gating. We found a cluster of sites in the N-terminal portion of the S4-S5 linker (D540, R541, Y542, E544) that, when replaced with a glycine residue, rescued the −50 mV shift caused by the G546L mutation restoring a WT-like voltage dependence of activation. None of these mutations affected the voltage dependence of activation in the WT background. All other mutations tested did not rescue WT function and presented the destabilized closed state phenotype that is characteristic of the G546L mutation. These results suggest that flexibility of the N-terminal S4-S5 linker contributes to stabilization of hERG channels in the closed state and that the native glycine, G546, affords this flexibility in WT channels.