Inhibition of K(Ca)2.2 and K(Ca)2.3 channel currents by protonation of outer pore histidine residues

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed K(Ca)2.2 (SK2) and K(Ca)2.3 (SK3) channel currents, with K(Ca)2.3 (pIC(50) of ∼6.8) being approximately fourfold more sensitive than K(Ca)2.2 (pIC(50) of ∼6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. K(Ca)2.2 and K(Ca)2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with K(Ca)2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of K(Ca)2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in K(Ca)2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape.