The recently resolved atomic structure of Xenopus KCNQ1 revealed a unique negatively charged extracellular protein surface (Fig. 2 A) surrounding the pore entrance of the channel (Sun and MacKinnon, 2017). Calculation of the electrostatic potential based on recent cryo-EM structures of KCNQ1 channels (Sun and MacKinnon, 2017, 2020) shows less electronegativity in the human ortholog (Fig. 2 A). Nevertheless, negatively charged side chains forming the surface of human KCNQ1, hypothetically, could account for K+o sensitivity by attracting K+ from the external medium. Such a postulation is in accordance with an earlier report showing a reduction in K+o sensitivity by mutation of the E290 residue (Wang et al., 2015). To understand more about the impact of an external negative potential on the inhibitory effects of K+o, we first generated a mutant in which all amino acids involved in the formation of the electronegative surface (E290, S291, E295, and D317) were exchanged for alanine. KCNQ1-specific current could be recorded neither from Xenopus oocytes injected with the corresponding mRNA nor from transfected HEK 293T cells (data not shown). The aspartic acid at the 317 position is strongly conserved among K+ channels (Fig. 2 A) suggesting that this residue is unlikely to be responsible for K+o sensitivity of the channel. Thus, we next investigated the E290A/S291A/E295A triple mutant. Marked reduction of current expression hampered the analysis of the concentration dependency in this channel. Nevertheless, the comparison of current amplitudes at 0.2 mM and 100 mM K+o concentrations demonstrated that K+o strongly inhibits the channel, the extent of which was even slightly (∼8%) higher than that of the wild type (Fig. 2 B). We next assessed the K+o sensitivity of corresponding single alanine mutants as well. The extent of inhibition of S291A and E295A mutants by external K+ was very similar to that of the triple mutant when current amplitudes at 0.2 mM and 100 mM K+o are compared (Fig. 2, C–E). The E290A mutation induced about 34% reduction in K+o-dependent inhibition relative to wild type (Fig. 2, C–E; and Table 1), which was consistent with an earlier study conducted in CHO cells with E290C mutant (Wang et al., 2015). We next introduced a positive charge at the E290 position by exchanging glutamic acid for arginine. The resultant channel exhibited marginally (∼6%) increased sensitivity to external K+ (Fig. 2, C and F). Neutralization of the E290 side chain by E290Q mutation, which preserves the side chain volume, produced a channel with K+o sensitivity similar to E290R, additionally causing a small leftward shift (ΔIC50 = 3.64 ± 1.96 mM) in the concentration–response curve (Fig. 2, C and F; and Table 1). Proline substitution of the E290 position created a channel with K+o sensitivity very similar to wild type (Fig. 2, C and F; and Table 1). These results suggest that the negative charge at the E290 position is not necessary for K+o sensitivity. The side-chain volume at the E290 position seems to be positively correlated with the extent of K+o-induced inhibition (Fig. 2 G). Remarkably, the E290W mutant with the largest possible side chain volume did not produce functional channels (data not shown). Xenopus oocytes injected with mRNA encoding the D317A mutant produced small currents that were not suitable for analysis, and proline, glycine, or tryptophan substitutions at this position did not result in functional expression. Altogether, these results indicate that the electronegative pore surface of KCNQ1 is not the cause of the K+o sensitivity of the channel. A slightly increased K+o sensitivity in S291A, E295A, E290R, E290Q, and E290A/S291A/E295A mutants as well as its decrease in E290A mutant is consistent with the general picture of the broad mutagenesis study described below, indicating that statistically significant changes in K+o sensitivity can be induced by mutations located in various regions of the channel pore.