K+-dependent gating of K(ir)1.1 channels is linked to pH gating through a conformational change in the pore

1.We have used giant patch-clamp recording to investigate the interaction b etween pH gating and K+-dependent gating in rat K(ir)1.1. (ROMK) channels h eterologously expressed in Xenopus oocytes. 2. Gating by intracellular protons (pH gating) and extracellular K+ ions (K (+)f-dependent gating) is a hallmark of K(ir)1.1. channels that mediate Ksecretion and control NaCl reabsorption in the kidney. pH gating is driven by protonation of an intracellular lysine residue (K80 in K(ir)1.1). K+-dep endent gating occurs upon withdrawal of K+ ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterica lly. 3. K+-dependent gating was shown to be strictly coupled to pH gating; it on ly occurred when channels were in the pH-inactivated closed state, but not in the open state. Moreover, K+-dependent gating was absent in the non-pH-g ated mutant K(ir)1.1(K80M). 4. Channels inactivated by K+-dependent gating were reactivated upon additi on of permeant ions to the extracellular side of the membrane, while imperm eant ions failed to induce channel reactivation. Moreover, mutagenesis iden tified two residues in the P-helix (L136 and V140 in K(ir)1.1) that are cru cial for K+-dependent gating. Replacement of these residues with the ones p resent in the non-K+-gated K(ir)2.1 abolished K+-dependent gating of K(ir)1 .1 channels without affecting pH gating. 5. The results indicate that pH gating and K+-dependent gating are coupled to each other via structural rearrangements in the inner pore involving the P-helix.