The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes

Abstract

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage-dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate

“…The S4 charge neutralization mutant R243A was previously shown to shift to more depolarized potentials and decrease the slope of the G(V) curve for KCNQ1 (14). We tested the hypothesis that the fluorescence signal, if it reports the movement of the voltage sensor, would change in a similar manner for this mutation.…”

“…To date, no gating current measurements have been reported for KCNQ1 channels, likely due to the low valence and slow movement of the KCNQ1 voltage sensor relative to other voltage-gated potassium channels (13,14). Previous efforts to characterize the KCNQ1 voltage sensor in the presence and absence of KCNE1 relied on cysteine accessibility studies, and although they were able to gain insights into the movements of the voltage sensor, they provide only a limited view of its behavior (15,16).…”

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

“…To more rigorously address the possibility that KCNQ1 channels can open before all four voltage sensors move, we explore another voltage sensor mutation, R231C, which leads to a constitutively active channel by locking the voltage sensor in the activated position (20,21). Again, using coexpression, we explore whether the presence of constitutively active voltage sensors within a coassembled channel of KCNQ1 and KCNQ1 R231C subunits generates voltage-independent (constitutive) current.…”

Steric hindrance between S4 and S5 of the KCNQ1/KCNE1 channel hampers pore opening

“…4, smaller delay in the mutants could be due to other reasons, for example, faster transition from the downstate to the pre-upstate. ARTICLE There have been several mutation studies on the S4 segment, mainly focusing on the role of the positively charged residues [30][31][32][33]43 . Wu et al 33 have shown that mutations on the first arginine (R1; Arg228) and the second arginine (R2; Arg231) of the S4 segment destabilize the closed state and mutations on the fourth arginine (R4; Arg237) and the six arginine (R6; Arg243) destabilize the open state.…”