title: Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells.
abstract: The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.
authors.affiliation: INSERM, U665, Paris, F-75015, France.
authors.forename: Nedjma
authors.initials: N
authors.lastname: Zidi-Yahiaoui
authors.forename: Isabelle
authors.initials: I
authors.lastname: Mouro-Chanteloup
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: D'Ambrosio
authors.forename: Claude
authors.initials: C
authors.lastname: Lopez
authors.forename: Pierre
authors.initials: P
authors.lastname: Gane
authors.forename: Caroline
authors.initials: C
authors.lastname: Le van Kim
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cartron
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin
authors.forename: Pierre
authors.initials: P
authors.lastname: Ripoche
mesh_terms: D000818:Animals; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004285:Dogs; D005434:Flow Cytometry; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008627:Mercuric Chloride / Q000494:pharmacology; D008744:Methylamines / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D027682:Cation Transport Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008744:Methylamines; D000644:Quaternary Ammonium Compounds; C422995:RHBG protein, human; C413213:RHCG protein, human; D008627:Mercuric Chloride; C027451:methylamine