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 between pH gating and K(+)-dependent gating in rat K(ir)1.1 (ROMK) channels heterologously expressed in Xenopus oocytes. 2. Gating by intracellular protons (pH gating) and extracellular K(+) ions (K(+)-dependent gating) is a hallmark of K(ir)1.1 channels that mediate K(+) secretion 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(+)-dependent gating occurs upon withdrawal of K(+) ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterically. 3. K(+)-dependent gating was shown to be strictly coupled to pH gating; it only 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-gated mutant K(ir)1.1(K80 M). 4. Channels inactivated by K(+)-dependent gating were reactivated upon addition of permeant ions to the extracellular side of the membrane, while impermeant ions failed to induce channel reactivation. Moreover, mutagenesis identified two residues in the P-helix (L136 and V140 in K(ir)1.1) that are crucial for K(+)-dependent gating. Replacement of these residues with the ones present 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.
Schulte
U
U
Department of Physiology II, University of Tübingen, Ob dem Himmelreich 7, 72074 Tübingen, Germany. uwe.schulte@uni-tuebingen.de
Weidemann
S
S
Ludwig
J
J
Ruppersberg
J
J
Fakler
B
B
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D004553:Electric Conductivity; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D007477:Ions; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000502:physiology*; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D014981:Xenopus
D007477:Ions; C510692:Kcnj1 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D006859:Hydrogen; D011188:Potassium