Mutation of nearby residues at LIN interaction sites displays lack of effect. (A) Concentration dependence of the LIN effect on G_max of indicated hKCNQ1 site I mutants. Data shown as mean ± SEM; n = 4–8 per data point. Concentration-response curves were fitted using Eq. 2 with the Hill coefficient constrained to 1 (see Materials and methods for details). Neither of the site I mutations for which G_max could be determined significantly altered the G_max effect induced by 70 µM of LIN compared with WT (P > 0.05 with one-way ANOVA followed by Dunnett’s multiple comparisons test). G_max for h279A could not be reliably determined due to unstable maximal conductance over time under control conditions. (B) Concentration dependence of the LIN effect on V₅₀ of indicated hKCNQ1 site II mutants. Data shown as mean ± SEM; n = 6–8 per data point. Concentration-response curves were fitted using Eq. 2 with the Hill coefficient constrained to −1 (see Materials and methods for details). The hL273W mutation did not affect the ability of LIN to shift V₅₀ (P > 0.05 with one-way ANOVA followed by Dunnett’s multiple comparisons test for 70 µM of LIN). The hA300W appeared to impair the ability of LIN to shift V₅₀, which was detected as an apparent lower LIN affinity for the V₅₀ effect; however, at 70 µM of LIN there was no difference compared with WT (P > 0.05 with one-way ANOVA followed by Dunnett’s multiple comparisons test). In our hands, none of the hL303F/W (xL293F/W), hL307W (xL297W), or hS330F/W (xS320F/W) mutants generated detectable K⁺ currents, and the hL307F (xL297F) mutant did not generate consistent K⁺ currents. (C) Side view of a LIN constant binder at site Iα and the S5 residue L261, which is near site I but predicted to not directly engage in LIN interaction. (D) Side view of a LIN constant binder at site II and the S5 residue I264, which is near site II but predicted to not directly engage in LIN interaction. (E) Experimental mutation of residues nearby to predicted binder residues does not impair the effect of LIN. Data shown as mean ± SEM; n = 4–5 per data point. Concentration-response curves were fitted using Eq. 2 with the Hill coefficient constrained to −1 (see Materials and methods for details). ΔV_50,max was constrained to shared values between WT and mutant to make the fits more robust, and data are shown as normalized effect (100% was defined as the maximal effect from the fit). For 70 µM LIN, ΔV₅₀ and ΔG_max were within ±1.5 mV and ±6%, respectively (P > 0.05 with one-way ANOVA followed by Dunnett’s multiple comparisons test to compare with WT). Rel., relative.