The outer pore histidine residue common to both K_Ca2.2 and K_Ca2.3 was crucial to block by [H⁺]_o. (A) Sequence alignment of predicted outer pore region that spans the S5-P helix-SF-S6 of K_Ca2.2 and K_Ca2.3, with the single H residue in K_Ca2.2 and the two H residues in the pore of K_Ca2.3 highlighted. (B) Schematic representation of the location of the highlighted residues in K_Ca2.2 (H337 and N368) and K_Ca2.3 (H491 and H522). (C) Homology model of the pore region S5-P helix-SF-S6 of K_Ca2.2 displaying two subunits of the channel tetramer with proposed positions of the H and N residues labeled and represented in space-filled mode. Representative (D) K_Ca2.2(H337N) and (E) K_Ca2.3(H491N) outside-out patch currents evoked by a voltage ramp from −100 to +100 mV. Both channel mutants were poorly sensitive to inhibition by extracellular pH 5.5. (F) The concentration–inhibition relationships of currents measured at −60 mV of K_Ca2.2(H337N), K_Ca2.3(H491N), and K_Ca2.2(H337N, N368H). The sensitivity of wild-type currents is shown for comparison.