title: Functional reconstitution into liposomes of purified human RhCG ammonia channel.
abstract: BACKGROUND
  Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties.
  
  
  RESULTS
  An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy.
  
  
  CONCLUSIONS
  This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.
authors.affiliation: INSERM UMR_S 665, Paris, France. isabelle.mouro-chanteloup@inserm.fr
authors.forename: Isabelle
authors.initials: I
authors.lastname: Mouro-Chanteloup
authors.forename: Sylvie
authors.initials: S
authors.lastname: Cochet
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Sandrine
authors.initials: S
authors.lastname: Genetet
authors.forename: Nedjma
authors.initials: N
authors.lastname: Zidi-Yahiaoui
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin
authors.forename: Olivier
authors.initials: O
authors.lastname: Bertrand
authors.forename: Pierre
authors.initials: P
authors.lastname: Ripoche
mesh_terms: D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism*; D001704:Biopolymers; D027682:Cation Transport Proteins / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005614:Freeze Fracturing; D006801:Humans; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009995:Osmosis; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D001704:Biopolymers; D027682:Cation Transport Proteins; D008081:Liposomes; D008562:Membrane Glycoproteins; D008744:Methylamines; C413213:RHCG protein, human; D011994:Recombinant Proteins; D000641:Ammonia; C027451:methylamine