Figure 5: Residue Y578 is a critical mediator of NAD⁺ inhibition.

(a) Depicts the relationship between helices D (light blue), F (blue), H (magenta), R (yellow) and CBS2 (red) in the inactivated ClC-1 model. The side chains of residues K195, E232 (E_ext), V292, Y578 (Y_cen) and D579, in addition to the backbone atoms of V292, are depicted as sticks coloured by atom type. Anion-binding sites S_ext (e) and S_int (i) are depicted as green spheres. Dashed lines represent possible polar interactions between residues. (b) Common gating curves for WT ClC-1 and Y578 mutants in the absence (open symbols) and presence (filled symbols) of 3 mM intracellular NAD⁺. All data are means and s.e.m. and solid lines are fits of equation (1) to the experimental data points. Dashed and dotted lines represent WT ClC-1 curves, as shown in the first figure of this panel, and are included for the purpose of comparison. (c) The mean shift of the voltage dependence of common gating curves (ΔV _1/2^common) by 3 mM intracellular NAD⁺. Data are means and linearly propagated values of s.e.m. For measurements with 3 mM NAD⁺ n=5 (WT ClC-1), 5 (Y578A), 5 (Y578F), 3 (Y578H) and 4 (Y578K) (separate experiments). Statistical significance (P<0.0001) was determined by one-way ANOVA.