Gating modulation of the KCNQ1 channel by KCNE proteins studied by voltage-clamp fluorometry

Figure 1. Structure and ionic currents of the KCNQ1 channel with KCNE1. (a) Top view (left) and side view (right) of the tetrameric KCNQ1 structures [37]. S1–S3, S4, S4–S5 linker, and S5–S6 are colored in green, red, orange and yellow, respectively. Dotted circles indicate putative KCNE1 sites. S4 segments (red helices) voltage-dependently move up and down as indicated by a cyan arrow in the right figure. (b) Representative currents and G-V curves of the KCNQ1 channel in Xenopus oocytes with or without KCNE1 co-expression. G/Gmax indicates apparent open probability of the channel. Co-expression of KCNE1 slows the activation of the channel and shifts the G-V curve in the positive direction (The channel requires higher membrane potential to be activated.). Inset shows the voltage clamp protocol. The currents at 0 mV are colored in red for comparison. Figures and the data presented are adapted from [26,40].

Figure 2. KCNE1 stabilizes the intermediate state of the KCNQ1 channel. (a) Preparation of fluorescence-labelled ion channels expressed in Xenopus oocytes for VCF experiments. Fluorescence changes are acquired by a photomultiplier tube (or a photodiode) while ionic currents are simultaneously recorded under two-electrode voltage clamp. (b) G-V (black; related to pore opening) and F-V (green; related to VSD movement) relationships of KCNQ1 alone and the KCNQ1-KCNE1 channel. The F-V relationship of KCNQ1-KCNE1 is split into two components, F1 and F2 [20]. (c) Model of three states of KCNQ1 channel gating without KCNE1 (upper row) and with KCNE1 (bottom row). Green circles indicate attached fluorophores (e.g., Alexa Fluor 488) for the VCF experiments. S4 segments (red) voltage-dependently move up and down (cyan arrows in upper row). KCNE1 stabilizes the intermediate state. Figures and the data presented are adapted from [26,40].

Figure 3. Bulky amino acid residues on S4 and S5 prevent the KCNQ1-KCNE1 channel from opening. (a) Side chain dependence of G-V curves (left) and voltage of half activation (V1/2, right) in KCNQ1 F232 mutant series. Mutation of a larger amino acid residue tends to require higher membrane potential to be activated. (b) Locations of F232 on S4 (red) and F279 on S5 (yellow) in the cryo-EM structure of the KCNQ1 channel [37]. S4 moves up and down in the direction of the cyan arrow. (c) Side chain dependence of G-V curves (left) and voltage of half activation (V1/2, right) in KCNQ1 F279 mutant series. As observed in the F232 mutants, mutation of a larger amino acid residue tends to require higher membrane potential to be activated. Figures and the data presented are adapted from [26,40].

Figure 4. F232 and F279 create a delay of channel opening after upward VSD movement. (a) Representative ionic currents (black traces) and fluorescence intensity (green traces) of KCNQ1 WT, F232A and F279A in the presence of KCNE1. Membrane potential was +40 mV from the holding potential of −90 mV. (b) F-V (green) and G-V relationships (black) of WT, F232A and F279A in the presence of KCNE1. (c) S4 segment (red bar) easily moves upward during depolarization in the absence of KCNE1. In the presence of KCNE1, the distance between S4 and S5 (yellow bar) becomes smaller and that may cause steric hindrance between the two phenylalanine residues. Figures and the data presented are adapted from [26,40].