K ⁺ _o sensitivity of alanine mutants spanning pore residues I268–V355. (A) Sample current traces from representative KCNQ1 channels in 0.2 mM and 100 mM [K⁺]_o. Dashed lines show 0 current. (B) Peak current analysis for representative channels shown in A normalized to the first point at 0.2 mM K⁺_o. Prolonged recordings at 100 mM K⁺_o is shown, n = 4–7, error bars represent ±SEM. (C) Summary of current amplitude changes in 100 mM [K⁺]_o relative to 0.2 mM [K⁺]_o for alanine mutants. n = 5–12, error bars represent ±SEM, one-way ANOVA, Dunnett’s post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001. Alanine mutants yielding no currents in oocytes are not shown. (D) Localization of residues, alanine mutation of which exert statistically significant changes compared with wild type on the recent cryo-EM structure of human KCNQ1 (PDB ID: 6UZZ; Sun and MacKinnon, 2020). The extent of mutation-induced changes, represented as color-coded arbitrary intensity, is calculated as ΔI_rel = [1 − I_100K Mut/I_0.2K Mut] – [1 − I_100K WT/I_0.2K WT]. Scale bar indicates ΔIrel. Inset shows a closer view of the region with a strong mutational effect.