Am J Physiol Cell Physiol AJP: Regulatory, Integrative and Comparative Physiology
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Am J Physiol Cell Physiol 297: C537-C547, 2009. First published June 24, 2009; doi:10.1152/ajpcell.00137.2009
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule

Nedjma Zidi-Yahiaoui,1,2,3 Isabelle Callebaut,4 Sandrine Genetet,1,2,3 Caroline Le Van Kim,1,2,3 Jean-Pierre Cartron,1,2,3 Yves Colin,1,2,3 Pierre Ripoche,1,2,3 and Isabelle Mouro-Chanteloup1,2,3

1Institut National de la Santé et de la Recherche Médicale, UMR-S665, 2Institut National de la Transfusion Sanguine, 3Université Paris Diderot-Paris 7, and 4Centre National de la Recherche Scientifique, UMR7590, Université Pierre et Marie Curie Paris 6, Paris France

Submitted 26 March 2009 ; accepted in final form 22 June 2009

Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH3 across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins 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, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH3 permeability of RhCG was determined and was found to be close to that of AmtB. With 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 was 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. The impact of the mutations 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.

ammonium transport; HEK cells; Rhesus family; pH variations; ammonia channel


Address for reprint requests and other correspondence: I. Mouro-Chanteloup, INSERM, UMR S 665, Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, Paris, F-75015, France (e-mail: isabelle.mouro-chanteloup{at}inserm.fr).






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