A Pair of Phenylalanine Residues on the S4 and S5 Segments Create a Physical and Energy Barrier for the Voltage Sensor in KCNQ1/KCNE1 Channel

Abstract

KCNQ1 channel, a voltage-gated potassium channel, and its auxiliary subunit KCNE1 form the slowly activating IKs channel, which underlies one of the major repolarizing currents in human heart. Several groups have reported that movement of the voltage sensor domain (VSD) is slowed by KCNE1, however, the molecular mechanism how KCNE1 slows VSD movement is still largely unknown. In the present study, we identified Phe232 on S4 segment is responsible for the G-V curve shift by KCNE1. We mutated Phe232 into various amino acid residues and found that Gibbs free energy of activation at 0 mV (ΔG0) of Phe232 mutants with KCNE1 showed a clear side chain volume dependence. This result implied that Phe232 might have to overcome a physical obstacle upon activation. We tried to identify a potential obstacle for Phe232 and found Phe279 on S5 segment as a candidate. Phe279 mutants with KCNE1 showed a similar side-chain-volume dependent ΔG0 change. Disulfide bond formation upon activation between F232C and F279C indicated that they are in close proximity during the course of activation. To confirm that Phe232 and Phe279 actually serve as obstacles during activation, we directly tracked the VSD movement by voltage clamp fluorometry by attaching Alexa 488 at G219C on the S3-S4 linker. As we expected, F232A actually accelerated the VSD movement. Although F279A did not significantly change the VSD movement, the upward movement of VSD immediately led the channel to the opening. Our present results suggest that the pair of phenylalanine residues create a physical and energy barrier for the VSD. This may be one of the major molecular mechanisms for the slow gating of KCNQ1/KCNE1 channels.