Regulation of K_ir Channels by Intracellular pH and Extracellular K⁺ : Mechanisms of Coupling

ROMK channels are regulated by internal pH (pH_i) and extracellular K⁺ (K⁺_o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H⁺, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K⁺_o, consistent with coupling between the responses to pH_i and K⁺_o. The ion selectivity sequence of the activation of the channel by cations was K⁺ ≅ Rb⁺ > NH₄⁺ >> Na⁺, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K⁺ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K⁺_o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K⁺_o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K⁺ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba²⁺ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H⁺. We conclude that decreasing pH_I increases the sensitivity of ROMK2 channels to K⁺_o by altering the properties of the selectivity filter.