Functional analysis of human RhCG: comparison with E. coli ammonium transporter reveals similarities in the pore and differences in the vestibule.
Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH(3) 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 NH(3) 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 NH(3) 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 NH(3) transport mechanisms.
Zidi-Yahiaoui
Nedjma
N
Institut National de la Santé et de la Recherche Médicale, UMR-S665, Paris, France.
Callebaut
Isabelle
I
Genetet
Sandrine
S
Le Van Kim
Caroline
C
Cartron
Jean-Pierre
JP
Colin
Yves
Y
Ripoche
Pierre
P
Mouro-Chanteloup
Isabelle
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D008562:Membrane Glycoproteins; D000644:Quaternary Ammonium Compounds; C413213:RHCG protein, human