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

Abstract

The delayed rectifier I Ks potassium channel, formed by coassembly of α-(KCNQ1) and β-(KCNE1) subunits, is essential for cardiac function. Although KCNE1 is necessary to reproduce the functional properties of the native I Ks channel, the mechanism(s) through which KCNE1 modulates KCNQ1 is unknown. Here we report measurements of voltage sensor movements in KCNQ1 and KCNQ1/KCNE1 channels using voltage clamp fluorometry. KCNQ1 channels exhibit indistinguishable voltage dependence of fluorescence and current signals, suggesting a one-to-one relationship between voltage sensor movement and channel opening. KCNE1 coexpression dramatically separates the voltage dependence of KCNQ1/KCNE1 current and fluorescence, suggesting an imposed requirement for movements of multiple voltage sensors before KCNQ1/KCNE1 channel opening. This work provides insight into the mechanism by which KCNE1 modulates the I Ks channel and presents a mechanism for distinct β-subunit regulation of ion channel proteins.

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

“…KCNQ1 with C214A/G219C/C331A mutations (which will be described as WT henceforth), the exactly same construct reported by Osteen et al 34 , was used as a background. In this construct, two endogenous cysteine residues, one located in the upper part of the S3 (Cys214) and the other located in the upper part of the S6 (C331), have been replaced with alanine to exclusively attach Alexa488 maleimide to G219C on the S3-S4 linker 34 . Neither these mutations nor the introduction of Alexa488 significantly affect the channel properties 34 .…”

“…In this construct, two endogenous cysteine residues, one located in the upper part of the S3 (Cys214) and the other located in the upper part of the S6 (C331), have been replaced with alanine to exclusively attach Alexa488 maleimide to G219C on the S3-S4 linker 34 . Neither these mutations nor the introduction of Alexa488 significantly affect the channel properties 34 . In the absence of KCNE1, WT construct showed fluorescence signal mostly when the ionic current was also evoked (Fig.…”

“…If Phe232 and Phe279 mutations truly change V 1/2 or the voltage sensor equilibrium, the voltage sensor movement might be changed in F232A and F279A mutants. We, therefore, applied VCF to directly track the voltage sensor movement in these mutants with simultaneous current recording 34,35,40 . KCNQ1 with C214A/G219C/C331A mutations (which will be described as WT henceforth), the exactly same construct reported by Osteen et al 34 , was used as a background.…”

“…We, therefore, applied VCF to directly track the voltage sensor movement in these mutants with simultaneous current recording 34,35,40 . KCNQ1 with C214A/G219C/C331A mutations (which will be described as WT henceforth), the exactly same construct reported by Osteen et al 34 , was used as a background. In this construct, two endogenous cysteine residues, one located in the upper part of the S3 (Cys214) and the other located in the upper part of the S6 (C331), have been replaced with alanine to exclusively attach Alexa488 maleimide to G219C on the S3-S4 linker 34 .…”

“…Other sitedirected mutagenesis studies also suggest that KCNE1 changes the VSD movement or conformation [30][31][32][33] . In addition, recent voltage clamp fluorometry (VCF) experiments clearly showed that KCNE1 alters VSD movement 34,35 . Based on these evidences, it is reasonable to think that KCNE1 slows the gating of KCNQ1, if not exclusively, via the VSD.…”