pH-dependent gating of ROMK (K(ir)1.1) channels involves conformational changes in both N and C termini

Abstract

ROMK channels (K(ir)1.1) are members of the superfamily of inward rectifier potassium channels (K(ir)) and represent the channels underlying K+ secretion in the kidney. As their native counterparts, K(ir)1.1 channels are gated by intracellular pH, with acidification leading to channel closure. Although a lysine residue (Lys80) close to the first hydrophobic segment M1 has been identified as the pH sensor, little is known about how opening and closing of the channel is accomplished. Here we investigate the gating process of K(ir)1.1 channels exploiting their state-dependent modification by water-soluble oxidants and sulfhydryl reagents. Mutagenesis of all intracellular cysteines either alone or in combination revealed two residues targeted by these reagents, one in the N terminus (Cys49) and one in the C terminus (Cys308) of the channel protein. Both sites reacted with the thiol reagents only in the closed state and not in the open state. These results indicate that pH-dependent gating of K(ir)1.1 channels involves movement of protein domains in both N and C termini of the K(ir)1.1 protein.