Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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

The rhesus protein RhCG: a new perspective in ammonium transport and distal urinary acidification

Urinary acidification is a complex process requiring the coordinated action of enzymes and transport proteins and resulting in the removal of acid and the regeneration of bicarbonate. Proton secretion is mediated by luminal H(+)-ATPases and requires the parallel movement of NH(3), and its protonation to NH(4)(+), to provide sufficient buffering. It has been long assumed that ammonia secretion is a passive process occurring by means of simple diffusion driven by the urinary trapping of ammonium. However, new data indicate that mammalian cells possess specific membrane proteins from the family of rhesus proteins involved in ammonia/μm permeability. Rhesus proteins were first identified in yeast and later also in plants, algae, and mammals. In rodents, RhBG and RhCG are expressed in the collecting duct, whereas in humans only RhCG was detected. Their expression increases with maturation of the kidney and accelerates after birth in parallel with other acid-base transport proteins. Deletion of RhBG in mice had no effect on renal ammonium excretion, whereas RhCG deficiency reduces renal ammonium secretion strongly, causes metabolic acidosis in acid-challenged mice, and impairs restoration of normal acid-base status. Microperfusion experiments or functional reconstitution in liposomes demonstrates that ammonia is the most likely substrate of RhCG. Similarly, crystal structures of human RhCG and the homologous bacterial AmtB protein suggest that these proteins may form gas channels.Kidney International advance online publication, 6 October 2010; doi:10.1038/ki.2010.386

Propriétés structurales et fonctionnelles des protéines RhBG et RhCG, transporteur d'ammonium chez les mammifères

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

TEM of negatively stained RhCG particles.

<p>The electron micrograph reflects the homogeneity of the purified protein. Scale bar is 50 nm. Arrows indicate some top-views of RhCG. In the insert, class averages corresponding to top-views of RhCG are displayed. Scale bar is 10 nm.</p

Ammonium ion transport by the AMT/Rh homologue LeAMT1;1

AMT (ammonium transporter)/Rh (Rhesus) ammonium transporters/channels are identified in all domains of life and fulfil contrasting functions related either to ammonium acquisition or excretion. Based on functional and crystallographic high-resolution structural data, it was recently proposed that the bacterial AmtB (ammonium transporter B) is a gas channel for NH(3) [Khademi, O'Connell, III, Remis, Robles-Colmenares, Miercke and Stroud (2004) Science 305, 1587–1594; Zheng, Kostrewa, Berneche, Winkler and Li (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 17090–17095]. Key residues, proposed to be crucial for NH(3) conduction, and the hydrophobic, but obstructed, pore were conserved in a homology model of LeAMT1;1 from tomato. Transport by LeAMT1;1 was affected by mutations of residues that were predicted to constitute the aromatic recruitment site for NH(4)(+) at the external pore entrance. Despite the structural similarities, LeAMT1;1 was shown to transport only the ion; each transported (14)C-methylammonium molecule carried a single positive elementary charge. Similarly, NH(4)(+) (or H(+)/NH(3)) was transported, but NH(3) conduction was excluded. It is concluded that related proteins and a similar molecular architecture can apparently support contrasting transport mechanisms

Stopped-flow analysis of water osmotic variations in liposomes and RhCG-proteoliposomes.

<p>Individual time courses of fluorescence quenching in RhCG-proteoliposomes were obtained at 20°C with empty liposomes and RhCG-proteoliposomes submitted to a 50 mosmol/kg H₂O osmotic gradient. Inserts represents the Arrhenius plots of temperature-dependent water permeation allowing the determination of activation energy (Ea) values from the slopes.</p

Stopped-flow analysis of ammonia (A) or methylamine (B) fluxes in liposomes and RhCG-proteoliposomes.

<p>Individual time courses of fluorescence change were obtained after submitting the empty liposomes and proteoliposomes containing purified RhCG protein to a 10 meq inwardly directed gradient of ammonium (A) or methylammonium (B). In (A) two different LPRs (Lipid/Protein ratio) were used: 150 and 300. Insert in A represents the Arrhenius plots of temperature-dependent ammonia permeation allowing the determination of activation energy (Ea) values from the slopes.</p

TEM of RhCG incorporated into liposomes.

<p>A, Cryo-electron microscopy of vitrified proteoliposomes. Arrows show unilamelar structures. Scale bar is 250 nm. B and C, Freeze fracture electron microscopy of empty liposomes and proteoliposomes (LPR 150) respectively. The arrows indicate RhCG particles. Scale bar is 250 nm.</p