Dimer interaction in the Hv1 proton channel

Figures

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  Fig. 1.. Identification of S1 interfacial residues with a functional cysteine scanning assay. (A) Principle of the perturbation scanning assay to identify interfacial positions in transmembrane α-helices. Positions facing a protein environment are depicted in red. Interfacial positions are circled in yellow. (B) Summary of the functional cysteine scanning assay. Residues whose cysteine mutation loses its effect with monomerization are highlighted in blue. Red squares represent residue positions for which cysteine mutation significantly perturbs channel function, either in the dimeric or monomeric background. We could not measure any proton current for either dimeric or monomeric channels containing the D160C mutation, so we considered that this mutation perturbed Hv1 function in both the dimer and the monomer. (C) Wengxiang diagram of S1 helix with residue positions color-coded as follows: Gray, low impact in both dimer and monomer; red, high impact in both dimer and monomer; pink, selectivity filter D160, also high impact in both dimer and monomer (no current); green, low impact in dimer and high impact in monomer; blue, high impact in dimer and low impact in monomer (putative…

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  Fig. 2.. Homology model of CiHv1 dimer. (A) Side view of the transmembrane segments of the CiHv1 dimer model. Transmembrane segments are depicted as follows: S1, orange; S2, blue; S3, green; and S4, red. Loops are depicted in white. Black rectangles delimit regions of interest at the level of the dimer interface. (B and C) CiHv1 dimer model viewed from its extracellular face. (B) Same color code as in A. (C) Each protomer is represented in a different color (cyan for protomer A and dark blue for protomer B) to highlight their relative arrangement. (D) Close-up view of the regions of interest highlighted in A. Same color code as in A for the transmembrane helices. The identity of the protomer (A or B) is indicated in parenthesis after the name of the transmembrane segment. S1 interfacial residues and their putative partners on the neighbor subunit are shown as sticks. In the Region II Inset, segments other than S4 (A) and S1 (B) have been removed for clarity. D160 is shown as pink sticks to mark the middle of the S1 segment. In the Region III Inset, the linker between S4 and the coiled-coil segment is colored in dark blue.

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  Fig. 3.. Validation by Western blot analysis of the transmembrane dimer interface. Immunoblots from Xenopus oocytes expressing hHv1*-I121C-GFP, hHv1*-A197C-GFP, or coexpressing hHv1*-I121C-GFP with untagged hHv1* (wt*) or hHv1*-A197C, and revealed with an anti-GFP antibody. Proteins were either purified from untreated [0 Cu(Phen)3] (Left) oocytes, or oocytes submitted to an oxidative treatment (Center and Right). In the latter case, purified proteins were either denatured in nonreducing [300 µM Cu(Phen)3] (Center) or reducing conditions [300 µM Cu(Phen)3 + 10% βME] (Right). The arrow indicates the heterodimer band formed by cross-linking of I121C and A197C. n.s., nonspecific band that is also present in oocytes that do not express hHv1 (uninjected condition). lad., ladder.

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  Fig. 4.. Steric hindrance between residues of the transmembrane dimer interface drives channel closure kinetics. (A and B) Shifts of V1/2 (ΔV1/2, A) and relative tail decay times (B) between WT channels and channels containing a cysteine mutation in S1 external half (S161 to I175). Superimposed in red are the theoretical orientation angles between the α-carbons of the given residue and S161 (taken as the reference), considering that these residues belong to an α-helix. Note that cysteine mutations at positions with orientations close to S161 specifically lead to leftward shifts of the G-V curve (ΔV1/2 < 0) and slowing of channel closure. (C) Representative current traces for a voltage step to +60 mV of oocytes expressing CiHv1 channels mutated at position L169 and superposition of their normalized tail currents. (D) Current traces of other Interface I residues (Upper) and superposition of the normalized WT and mutant tail currents (Lower). (E–H) Relative tail decay times as a function of the volume of the residue (50) introduced at positions S161 (E), V165 (F), L169 (G), and K173 (H). Note the linear relationship (black line) between the log of the relative tail decay time and the volume…

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  Fig. 5.. Putative partners of S1 interfacial residues on S4 also exert a strong control on channel closure. (A) Representative current traces for a voltage step to +60 mV of oocytes expressing CiHv1 channels containing a cysteine mutation at S4 positions L250, I253, and L256. (B) Superposition of the normalized tail currents of WT (black), L250C (blue), and L256C (purple) channels. Note the slowing of the tail currents compared to WT channels. (C) Superposition of the normalized tail currents of WT, S161C (red), I253C (green), and S161C-I253C (yellow) channels from a +20 to −120 mV voltage step. (D) Relative tail decay times of S161C (n = 6), I253C (n = 8), and S161C-I253C (n = 7) mutants. The hashed bar indicates the theoretical tail decay time of S161C-I253C mutant, had the two mutations been independent.