Submitted on March 26, 2009
Revised on June 12, 2009
Accepted on June 22, 2009
Functional analysis of human RhCG: a comparison with E.coli ammonium transporter reveals similarities in the pore and differences in the vestibule
Nedjma Zidi-Yahiaoui1, Isabelle Callebaut2, Sandrine Genetet1, Caroline Le Van Kim1, Jean-Pierre Cartron1, Yves Colin1, Pierre Ripoche1, and Isabelle Mouro-Chanteloup1*
1 INSERM, UMR-S 665
2 Université Pierre et Marie Curie
* To whom correspondence should be addressed. E-mail: isabelle.mouro-chanteloup{at}inserm.fr.
Rh glycoproteins are members of the Amt/Mep/Rh family facilitating movement of NH3 across plasma membranes. Homology models constructed on the basis of the experimental structures of E.coli AmtB and N.europaea Rh50 indicated a channel structure for human RhAG, RhBG, RhCG in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, "twin-Phe". In this study, the RhCG function was investigated by stopped-flow measuring kinetic pH-variations in HEK293E cells in the presence of ammonium gradient. The apparent unitary NH3 permeability of RhCG was determined and was found to be close to that of AmtB. Using a site-directed mutagenesis approach, critical residues involved in Rh NH3 channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid were shown. In contrast to AmtB, individual mutations of each phenylalanine of the "twin-Phe" impaired the function while the removal of both resulted in recovery of the transport activity. Mutations impact suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the NH3 transport mechanisms.
