Human Rhesus-associated glycoprotein mediates facilitated transport of NH ₃ into red blood cells

Abstract

ammonium ͉ mouse mutants ͉ Rhesus deficiency

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells

“…Secondly, the significant decrease in E a associated with RhBG and RhCG expression also points towards RhBG and RhCG as channels rather than as active exchangers whose function requires high-energy-dependent conformational changes [28,29]. The conclusion that RhBG and RhCG carry out NH 3 transport fully correlates with our recent study demonstrating that the rate constant of NH 3 movement in RBCs is strictly dependent on RhAG expression level [20]. These results, supported by crystallographic and functional studies demonstrating that AmtB, a bacterial member of the Amt/Mep/Rh superfamily, is a channel that conducts NH 3 [30,31], first identified Rh glycoproteins as members of an NH 3 channel family in mammals.…”

“…This method permits reliable measurement of rapid kinetics in a fraction-of-a-second range and is therefore particulary suitable if NH 3 is the transported species. The intracellular alkalinization and acidification processes observed in the presence of inwardly and outwardly ammonium gradients, respectively, could be explained by NH 4 + /H + exchange, as proposed by others [6,15,17] or by facilitated NH 3 transport, as has been proposed for the erythroid Rh glycoprotein RhAG [20]. However, two arguments prompt us to favour the hypothesis that the uncharged molecule is transported: an influx of NH 3 resulting in the intracellular captation of H + to give NH 4 + , and an efflux of NH 3 resulting from deprotonation of NH 4 + leaving one intracellular H + ion behind [26].…”

“…Although RhAG was shown to facilitate NH 3 movement across the RBC membrane [20], the transport function of this Rh glycoprotein could not be assessed in the cellular model succesfully used for RhBG and RhCG. Of note, the expression level of RhAG in transfected HEK-293 cells (9500 copies of RhAG/ cell) is lower than that observed in Rh null RBCs of the amorph type (17 000 copies of RhAG/RBC) which already exhibited much reduced alkalinization rate constant values, as compared with normal control RBCs (100 000 copies of RhAG/cell) [20].…”

“…Using stopped-flow fluorimetry, we have recently defined the ammonium transport characteristics of RhAG in RBCs from human and mouse genetic variants that exhibit quantitatively different expression of RhAG. The kinetic rates of pH i (intracellular pH) changes in resealed ghost cells submitted to inwardly directed methylammonium or ammonium gradients, and the ammonium permeability of the channel unit strongly suggested that RhAG facilitates NH 3 movement across the RBC membrane [20]. Here, we used the stopped-flow based approach to analyse the characteristics of ammonium transport mediated by RhBG and RhCG in transfected kidney cells.…”

Evidence of a Structural and Functional Ammonium Transporter RhBG·Anion Exchanger 1·Ankyrin-G Complex in Kidney Epithelial Cells

“…By its association with proteins Rh, the carrier of the Rhesus blood group antigens, and RhAG, which define the core of the Rh membrane complex, eAE1 modulates their expression level (4). RhAG belongs to the ammonium transporters/methylammonium-ammonium permease/Rh superfamily of ammonium transporters and allows the facilitated transport of NH 3 across the RBC membrane (5). Ankyrin-R mediates skeleton attachment of Rh and RhAG (6); therefore an Rh⅐eAE1 macrocomplex model has been proposed (4) in which the Rh complex and eAE1 are associated with each other and the spectrin-based skeleton through their common direct interaction with ankyrin-R (6).…”