Functional reconstitution into liposomes of purified human RhCG ammonia channel.

<h4>Background</h4>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...

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Autores principales:	Isabelle Mouro-Chanteloup, Sylvie Cochet, Mohamed Chami, Sandrine Genetet, Nedjma Zidi-Yahiaoui, Andreas Engel, Yves Colin, Olivier Bertrand, Pierre Ripoche
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/90d1b473c11c47b6bd85545c6ae18a63
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spelling	oai:doaj.org-article:90d1b473c11c47b6bd85545c6ae18a632021-11-25T06:26:16ZFunctional reconstitution into liposomes of purified human RhCG ammonia channel.1932-620310.1371/journal.pone.0008921https://doaj.org/article/90d1b473c11c47b6bd85545c6ae18a632010-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20126667/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.Isabelle Mouro-ChanteloupSylvie CochetMohamed ChamiSandrine GenetetNedjma Zidi-YahiaouiAndreas EngelYves ColinOlivier BertrandPierre RipochePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 1, p e8921 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Isabelle Mouro-Chanteloup Sylvie Cochet Mohamed Chami Sandrine Genetet Nedjma Zidi-Yahiaoui Andreas Engel Yves Colin Olivier Bertrand Pierre Ripoche Functional reconstitution into liposomes of purified human RhCG ammonia channel.
description	<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
format	article
author	Isabelle Mouro-Chanteloup Sylvie Cochet Mohamed Chami Sandrine Genetet Nedjma Zidi-Yahiaoui Andreas Engel Yves Colin Olivier Bertrand Pierre Ripoche
author_facet	Isabelle Mouro-Chanteloup Sylvie Cochet Mohamed Chami Sandrine Genetet Nedjma Zidi-Yahiaoui Andreas Engel Yves Colin Olivier Bertrand Pierre Ripoche
author_sort	Isabelle Mouro-Chanteloup
title	Functional reconstitution into liposomes of purified human RhCG ammonia channel.
title_short	Functional reconstitution into liposomes of purified human RhCG ammonia channel.
title_full	Functional reconstitution into liposomes of purified human RhCG ammonia channel.
title_fullStr	Functional reconstitution into liposomes of purified human RhCG ammonia channel.
title_full_unstemmed	Functional reconstitution into liposomes of purified human RhCG ammonia channel.
title_sort	functional reconstitution into liposomes of purified human rhcg ammonia channel.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/90d1b473c11c47b6bd85545c6ae18a63
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Functional reconstitution into liposomes of purified human RhCG ammonia channel.

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