By examining the consequences both of changes of [K+]o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni2+ is similar to that for proton block. Ni2+ block is inhibited by increasing [K+]o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni2+ had no effect on gating currents. We propose that, as with Ho+, Ni2+ binding to H463 facilitates an outer pore inactivation process that is antagonized by Ko+ and that requires R487. However, whereas Ho+ substantially accelerates inactivation of residual currents, Ni2+ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co2+ and Mn2+, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0mM Ko+ is Zn2+ (KD∼0.07mM)≥Ni2+ (KD∼0.15mM)>Co2+ (KD∼1.4mM)>Mn2+ (KD>10mM).

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn2+ to inhibit macroscopic hKv1.5 currents, and using site‐directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H+o and Zn2+. As with Zn2+, H+o caused a concentration‐dependent, K+o‐sensitive and reversible reduction of the maximum conductance (gmax). With zero, 5 and 140 mm K+o the pKH for this decrease of gmax was 6.8, 6.2 and 6.0, respectively. The concentration dependence of the block relief caused by increasing [K+]o was well fitted by a non‐competitive interaction between H+o and K+o, for which the KD for the K+ binding site was 0.5‐1.0 mm. Additionally, gating current analysis in the non‐conducting mutant hKv1.5 W472F showed that changing from pH 7.4 to pH 5.4 did not affect Qmax and that charge immobilization, presumed to be due to C‐type inactivation, was preserved at pH 5.4. Inhibition of hKv1.5 currents by H+o or Zn2+ was substantially reduced by a mutation either in the channel turret (H463Q) or near the pore mouth (R487V). In light of the requirement for R487, the homologue of Shaker T449, as well as the block‐relieving action of K+o, we propose that H+ or Zn2+ binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.