title: Mechanism of proton gating of a urea channel.
abstract: The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [(14)C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.
authors.affiliation: Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Weeks
authors.forename: Gene
authors.initials: G
authors.lastname: Gushansky
authors.forename: David R
authors.initials: DR
authors.lastname: Scott
authors.forename: George
authors.initials: G
authors.lastname: Sachs
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D044923:Helicobacter hepaticus / Q000378:metabolism; D016480:Helicobacter pylori / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D012313:RNA / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000378:metabolism; D014508:Urea / Q000737:chemistry* / Q000378:metabolism; D014510:Urease / Q000378:metabolism; D014981:Xenopus; D014982:Xenopus laevis
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011522:Protons; C114527:UreI protein, Helicobacter pylori; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D012313:RNA; D014508:Urea; D014510:Urease