title: Subunit-subunit interactions are critical for proton sensitivity of ROMK: evidence in support of an intermolecular gating mechanism.
abstract: The tetrameric K channel ROMK provides an important pathway for K secretion by the mammalian kidney, and the gating of this channel is highly sensitive to changes in cytosolic pH. Although charge-charge interactions have been implicated in pH sensing by this K channel tetramer, the molecular mechanism linking pH sensing and the gating of ion channels is poorly understood. The x-ray crystal structure KirBac1.1, a prokaryotic ortholog of ROMK, has suggested that channel gating involves intermolecular interactions of the N- and C-terminal domains of adjacent subunits. Here we studied channel gating behavior to changes in pH using giant patch clamping of Xenopus laevis oocytes expressing WT or mutant ROMK, and we present evidence that no single charged residue provides the pH sensor. Instead, we show that N-C- and C-C-terminal subunit-subunit interactions form salt bridges, which function to stabilize ROMK in the open state and which are modified by protons. We identify a highly conserved C-C-terminal arginine-glutamate (R-E) ion pair that forms an intermolecular salt bridge and responds to changes in proton concentration. Our results support the intermolecular model for pH gating of inward rectifier K channels.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026, USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Leng
authors.forename: Gordon G
authors.initials: GG
authors.lastname: MacGregor
authors.forename: Ke
authors.initials: K
authors.lastname: Dong
authors.forename: Gerhard
authors.initials: G
authors.lastname: Giebisch
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hebert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D015640:Ion Channel Gating; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011522:Protons; D016415:Sequence Alignment; D014982:Xenopus laevis
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D020033:Protein Isoforms; D021122:Protein Subunits; D011522:Protons; D001120:Arginine; D008239:Lysine