Moving the pH Gate of the Kir1.1 Inward Rectifier Channel

Abstract

Both structural and functional studies suggest that pH gating of the inward rectifier potassium (K) channel, Kir1.1 (ROMK), is mediated by the convergence of 4 hydrophobic leucines (one from each subunit) near the cytoplasmic bundle-crossing of the inner transmembrane helices. We tested this hypothesis by moving the putative leucine gate from the L160-Kir1.1b to other positions along the inner transmembrane helix, and measuring inward current and conductance as functions of internal pH, using the Xenopus oocyte heterologous expression system. Results of these studies indicated that it was possible to replace the putative inward rectifier pH gate at L160-Kir1.1b by either a leucine or methionine at 157-Kir1.1b (G157L-L160G or G157M-L160G). Although both leucine and methionine gated the channel at 157-Kir1.1b, residues of similar hydrophobicity (tyrosine and valine) did not. Hence, hydrophobicity was a necessary but not a sufficient condition for steric gating at 157. This was in contrast to the 160-Kir1.1b locus, where side-chain hydrophobicity was both a necessary and sufficient property for steric gating. Homology models were constructed for all mutants that expressed significant whole-cell currents, using the closed-state coordinates of the prokaryotic inward rectifier, KirBac1.1. Models of mutants that retained pH gating were too narrow at the bundle crossing to permit hydrated K ion permeation in the closed-state. On the other hand, mutants that lost pH gating had ample space at the bundle crossing for hydrated K permeation in the closed-state. These results support our hypothesis that hydrophobic leucines at the cytoplasmic end of the inner transmembrane helices comprise the principal pH gate of Kir1.1, a gate that can be relocated from 160-Kir1.1b to 157-Kir1.1b.