title: Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori.
abstract: Helicobacter pylori's unique ability to colonize and survive in the acidic environment of the stomach is critically dependent on uptake of urea through the urea channel, HpUreI. Hence, HpUreI may represent a promising target for the development of specific drugs against this human pathogen. To obtain insight into the structure-function relationship of this channel, we developed conditions for the high-yield expression and purification of stable recombinant HpUreI. Detergent-solubilized HpUreI forms a homotrimer, as determined by chemical cross-linking. Urea dissociation kinetics of purified HpUreI were determined by means of the scintillation proximity assay, whereas urea efflux was measured in HpUreI-containing proteoliposomes using stopped-flow spectrometry to determine the kinetics and selectivity of the urea channel. The kinetic analyses revealed that urea conduction in HpUreI is pH-sensitive and saturable with a half-saturation concentration (or K(0.5)) of ~163 mM. The extent of binding of urea by HpUreI was increased at lower pH; however, the apparent affinity of urea binding (~150 mM) was not significantly pH-dependent. The solute selectivity analysis indicated that HpUreI is highly selective for urea and hydroxyurea. Removing either amino group of urea molecules diminishes their permeability through HpUreI. Similar to urea conduction, diffusion of water through HpUreI is pH-dependent with low water permeability at neutral pH.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City, Iowa 52241, United States.
authors.forename: Lawrence R
authors.initials: LR
authors.lastname: Gray
authors.forename: Sean X
authors.initials: SX
authors.lastname: Gu
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Shahram
authors.initials: S
authors.lastname: Khademi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D016480:Helicobacter pylori / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D014508:Urea / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; C114527:UreI protein, Helicobacter pylori; D014508:Urea