Mutations Within the S4-S5 Linker Alter Voltage Sensor Constraints During Activation and Deactivation of Herg K+ Channels

Abstract

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.