Inhibition of outward KCNQ1 current is induced by external permeant ions. (A) Sample current traces of KCNQ1 channels at 100 mM external monovalent cations as indicated. Currents were continuously recorded at 120-s interpulse intervals. Representative traces correspond to second point of each type of the monovalent ion shown in B. Dashed lines represent 0 current. (B) Changes of outward peak current amplitude at +60 mV upon sequential substitution of external monovalent cations normalized to amplitudes of the first point recorded in external 100 mM Na⁺, n = 5–8, error bars represent ±SEM. Lines are the fits of the G269A and F351A data to linear function; F351AK⁺ = 0.374 ± 0.003; F351ARb⁺ = 0.282 ± 0.003; G269AK⁺ = 0.701 ± 0.002; G269ARb⁺ = 0.697 ± 0.003, ***P = 0.0007; n.s., nonsignificant (ANCOVA linear regression analysis). (C) Representative current traces from recordings of the F340A mutant in the presence of various external monovalent cations (100 mM). The corresponding analysis is shown on the right side of the traces. n = 5, error bars represent ±SEM. (D) Current traces and the corresponding analysis of F340A mutant when the sequence of solution exchange was modified as indicated; n = 4, error bars represent ±SEM. (E) Example of current traces mediated through F351A mutant at five different concentrations of external K⁺ and Rb⁺ as indicated. (F) Peak current inhibition for F351A in external K⁺ and Rb⁺ conditions normalized to their values at 0.2 mM, n = 8 for Rb⁺_o and n = 10 for K⁺_o, error bars represent ±SEM. (G) Concentration–response curves after subtraction of theoretical values according to the GHK equation. Solid curves represent fits of the data to the Hill equation. The parameters of inhibition are shown in Table 1. Error bars represent ± SEM.