Rocheleau JM , Gage SD , Kobertz WR .

GM0707650 NIGMS NIH HHS , R01 GM070650 NIGMS NIH HHS , R01 GM070650-02 NIGMS NIH HHS

	Figure 1. KCNE1 alanine mutants show diverse gating properties. (A) Two-electrode voltage clamp recordings of WT, H73A, and E83A mutant channels expressed in Xenopus oocytes. Currents were recorded in KD98 solution. Dashed line indicates zero current. Scale bars represent 1 μA and 0.5 s. Inset, protocol of 4-s depolarizations from −80 to 60 mV at 10-mV increments used to elicit currents shown. (B) Voltage activation curves for WT and representative mutant channel complexes calculated from tail current analysis. Solid curves represent Boltzmann fits to the data. Data was averaged from 5–10 oocytes each ± SEM.
	Figure 2. Periodicity of gating perturbations in the KCNE1 C-terminal domain. Center, helical wheel diagram of the 19 C-terminal E1 residues examined. Red circles indicate residues with ΔΔGiso > 1 kcal/mol, blue circles indicate ΔΔGiso ≤ 1 kcal/mol. Power spectrum analysis indicates a peak angle of 125° and α-PI of 1.77 for the entire C-terminal segment. When residues above and below P77 are plotted on separate helical wheels (left and right), high impact residues (red) and low impact residues (blue) segregate to separate faces of each helical diagram. Each high impact face is denoted by a red line. S68A is a high impact residue on the low impact face (filled blue with red outline). D76A is a nonfunctional mutant at the plasma membrane, as determined by cell surface luminometry (red fill).
	Figure 3. Negatively charged side chains produce smaller perturbations than alanine at position S68. (A) TEVC recordings of S68A and S68D channels expressed in Xenopus oocytes. Currents were recorded in KD98 solution. Dashed line indicates zero current. Scale bars represent 1 μA and 0.5 s. Inset, protocol of 4-s depolarizations from −80 to 60 mV at 10-mV increments used to elicit currents shown. (B) Voltage activation curves for S68A, S68D, and S68E mutant channels calculated from tail current analysis. Solid curves represent Boltzmann fits to the data. Dotted line indicates Boltzmann fit of WT activation curve. Data was averaged from 8–10 oocytes each ± SEM.
	Figure 4. Branched amino acids cause larger perturbations at position S74. (A) TEVC recordings of S74L, S74M, and S74I channels expressed in Xenopus oocytes. Currents were recorded in KD98 solution. Dashed line indicates zero current. Scale bars represent 1 μA and 0.5 s. Inset, protocol of 4-s depolarizations from –80 to 60 mV at 10-mV increments used to elicit currents shown. (B) Voltage activation curves for S74 mutant channels calculated from tail current analysis. Solid curves represent Boltzmann fits to the data. Dotted line indicates Boltzmann fit of WT activation curve. Data was averaged from 6–10 oocytes each ± SEM.
	Figure 5. The transmembrane-abutting C-terminal domain is comprised of two helical regions. (A) Whole oocyte luminometry was used to quantify the surface expression of HA-tagged E1 peptides (E1, D76N, D76A) and uninjected controls (UN). Luminescence is reported in relative light units (RLU). Error bars represent SEM from 10–20 oocytes. (B) Periodicity analysis of the top (K69–D76) and bottom (P77–A86) segments of the E1 C-terminal domain. P(ω) is plotted as a function of angular frequency (ω) to generate a power spectrum of the ΔΔGiso values for each segment. A value of 1 kcal/mol was assigned for the nonfunctional D76A mutant. The primary peak occurs at 114° for the top segment and 112° for the bottom. (C) A cartoon of the cytoplasmic E1 C-terminal domain split into two helical regions by a kink or turn. (D) Changes in deactivation rates mirror isochronal ΔΔG measurements for the E1 C-terminal domain mutants. Double bar graph comparing ΔΔGiso and deactivation rates (τd) for the E1 C-terminal alanine and leucine mutants. Solid bars indicate ΔΔGiso, open bars \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}_{{\mathrm{d}}}^{{\mathrm{WT}}}/{\tau}_{{\mathrm{d}}}^{{\mathrm{mutant}}}\end{equation*}\end{document}. The dashed denotes the cutoff values for ΔΔGiso and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\tau}_{{\mathrm{d}}}^{{\mathrm{WT}}}/{\tau}_{{\mathrm{d}}}^{{\mathrm{mutant}}}\end{equation*}\end{document}.

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