Substitution Scan of the S4-S5 Linker Region in KCNQ1 Channel: Structural Scaffold for Critical Protein Interactions

Abstract

KCNQ1 α-subunits are composed out of six transmembrane segments (S1-S6) that tetramerize into a functional channel. In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac IKs and consequently mutations in KCNQ1 are linked to the congenital LQT1 syndrome. Similar to other Kv channels, the S1-S4 segments form the voltage sensing domain that senses the membrane potential and that controls the opening or closure of the channel gate through an electromechanical coupling. In other channels a direct interaction between the S4-S5 linker and bottom part of S6 has been shown to constitute this electromechanical coupling. We previously identified residues in the C-terminal part of S6 that are critical for KCNQ1 gating. To investigate if these residues interact with the S4-S5 linker, we performed an alanine/tryptophan substitution scan of the S4-S5 linker sequence. Based on their impact on channel gating, we categorized these substitutions as either “high” or “low impact”. The pattern of “high impact” positions was consistent with an α-helical configuration and clustered on one side of the S4-S5 linker. Since substitutions at these positions markedly impaired channel gating, they are good candidates to contact residues in the bottom part of S6. Indeed, replacing valine 254 in the S4-S5 linker by a leucine resulted in channels that were partially constitutively open but channel closure could be rescued by combining V254L with the S6 mutation L353A that by itself displayed a similar phenotype as V254L. The observation that all known LQT1 mutations in the S4-S5 linker map on the “high impact” side further strengthens the proposal that this face of the S4-S5 linker contacts the C-terminal S6 segment and constitutes part of the electromechanical coupling in KCNQ1 channels.