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Identification of the Erythrocyte Rh Blood Group Glycoprotein as a Mammalian Ammonium Transporter

2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês

10.1074/jbc.c200060200

1083-351X

Connie M. Westhoff, Michelle Ferreri-Jacobia, Don‐On Daniel Mak, J. Kevin Foskett,

Neonatal Health and Biochemistry

The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopusoocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [14C]methylamine (MA), had an apparent EC50 of 1.6 mm and a maximum uptake rate (V max) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na+ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H+ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC50 = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H+-coupled counter-transport mechanism. The Rh blood group proteins are well known as the erythrocyte targets of the potent antibody response that causes hemolytic disease of the newborn. These proteins have been described in molecular detail; however, little is known about their function. A transport function is suggested by their predicted structure and from phylogenetic analysis. To obtain evidence for a role in solute transport, we expressed Rh proteins in Xenopusoocytes and now demonstrate that the erythroid Rh-associated glycoprotein mediates uptake of ammonium across cell membranes. Rh-associated glycoprotein carrier-mediated uptake, characterized with the radioactive analog of ammonium [14C]methylamine (MA), had an apparent EC50 of 1.6 mm and a maximum uptake rate (V max) of 190 pmol/oocyte/min. Uptake was independent of the membrane potential and the Na+ gradient. MA transport was stimulated by raising extracellular pH or by lowering intracellular pH, suggesting that uptake was coupled to an outwardly directed H+ gradient. MA uptake was insensitive to additions of amiloride, amine-containing compounds tetramethyl- and tetraethylammonium chloride, glutamine, and urea. However, MA uptake was significantly antagonized by ammonium chloride with inhibition kinetics (IC50 = 1.14 mm) consistent with the hypothesis that the uptake of MA and ammonium involves a similar H+-coupled counter-transport mechanism. Rh-associated glycoprotein methylamine The human Rh blood group proteins have been known for decades to cause hemolytic disease of the newborn, which can result in severe fetal morbidity and mortality (1.Race R.R. Sanger R. Blood Groups in Man. 6th Ed. Blackwell, Oxford1975Google Scholar). Despite their clinical importance, these multipass membrane proteins were not successfully isolated until the late 1980s (2.Agre P. Saboori A.M. Asimos A. Smith B.L. J. Biol. Chem. 1987; 262: 17497-17503Abstract Full Text PDF PubMed Google Scholar), and little progress was made in their characterization until the genes were cloned (3.Cherif-Zahar B. Bloy C. Le Van Kim C. Blanchard D. Bailly P. Hermand P. Salmon C. Cartron J.-P. Colin Y. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6243-6247Crossref PubMed Scopus (258) Google Scholar). Database searches for protein sequences with similarities to Rh proteins were not informative until the Caenorhabditis elegans sequencing initiative revealed that homologs exist in that species. These homologs, in turn, revealed a distant similarity between Rh proteins and ammonium transporters from bacteria and yeast. Since then additional Rh homologs have been found in many organisms (4.Kitano T. Saitou N. Immunogenetics. 2000; 51: 856-862Crossref PubMed Scopus (27) Google Scholar), and nonerythroid Rh homologs were detected in human and mouse kidney, testis, brain, and liver (5.Liu Z. Peng J. Mo R. Hui C.C. Huang C.H. J. Biol. Chem. 2001; 276: 1424-1433Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,6.Liu Z. Chen Y. Mo R. Chi-chung H. Cheng J.F. Mohandas N. Huang C.H. J. Biol. Chem. 2000; 275: 25641-25651Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The erythrocyte Rh blood group antigens are carried on two 417-amino acid polypeptides, RhD and RhCE, which are 97% identical. Rh-negative individuals carry a deletion or mutation in RHD and lack RhD protein (for reviews, see Refs. 7.Avent N.D. Reid M.E. Blood. 2000; 95: 375-387Crossref PubMed Google Scholar and 8.Huang C.H. Liu P.Z. Chen J.G. Semin. Hematol. 2000; 37: 150-165Crossref PubMed Scopus (93) Google Scholar). A related 409-amino acidRh-associated glycoprotein (RhAG)1 shares 37% amino acid identity with RhD and RhCE. RhAG and the RhD and RhCE proteins are predicted to span the membrane 12 times and associate in heterotetramers (9.Ridgwell K. Eyers S.A.C. Mawby W.J. Anstee D.J. Tanner M.J.A. J. Biol. Chem. 1994; 269: 6410-6416Abstract Full Text PDF PubMed Google Scholar). RhAG is important for the trafficking of RhD and RhCE to the plasma membrane as mutations in RHAG, independent of RH, result in Rhnullerythrocytes, which lack RhAG, RhD, and RhCE (8.Huang C.H. Liu P.Z. Chen J.G. Semin. Hematol. 2000; 37: 150-165Crossref PubMed Scopus (93) Google Scholar). Rhnullerythrocytes have structural defects, characterized morphologically as stomatocytes and spherocytes, as well as cation content abnormalities. Affected individuals have a compensated hemolytic anemia (10.Ballas S. Clark M.R. Mohandas N. Colfer H.F. Caswell M.S. Bergen M.O. Perkins H.A. Shohet S.B. Blood. 1984; 63: 1046-1055Crossref PubMed Google Scholar, 11.Sturgeon P. Blood. 1970; 36: 310-320Crossref PubMed Google Scholar). The low levels of endogenous expression of Rh proteins in mammalian cells, and the inability to express sufficient levels of recombinant forms, have hampered functional studies. Weak complementation by Rh proteins of a yeast ammonium mutant (12.Marini A.M. Matassi G. Raynal V. Andre B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (303) Google Scholar) has been criticized because ammonium uptake was not detected, and indirect evidence suggested that RhAG was involved in export, which contradicted the complementation results (13.Soupene E. Ramirez R.M. Kustu S. Mol. Cell. Biol. 2001; 21: 5733-5741Crossref PubMed Scopus (77) Google Scholar). We have used the Xenopus oocyte expression system to determine the function of Rh and RhAG proteins. Here we demonstrate successful expression of fully glycosylated RhAG protein and provide the first direct evidence for RhAG-mediated ammonium uptake. Human RhAG and RhCE cDNAs were obtained by PCR from reticulocyte RNA. cDNAs were cloned into a pSP64 vector (provided by B. Skach, Oregon Health Sciences University, Portland, OR) or the pBF vector (provided by F. Ashcroft, Oxford, UK) for transcripts with a poly(A) tail. Capped cRNA was synthesized with SP6 RNA polymerase from plasmid DNA with mMessage mMachine (Ambion, Inc., Austin, TX). Stage V and VI defolliculated oocytes were injected with 34 nl (1 ng/nl) of cRNA, or water for controls, and placed in individual wells in 96-well plates containing 200 μl of SOS in mm(100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6, 200 mosm) with 2.5 mm sodium pyruvate, 100 μg/ml gentamicin, and 100 μm N-acetyl-Leu-Leu-norleucinal at 16 °C. Radiolabeled [14C]methylammonium (CH3NH3 +) (ICN, Irvine, CA) uptake was measured 3 days postinjection. Experiments were performed at room temperature by placing groups of six to eight oocytes in 200 μl of low K+ (0.2 mm) SOS uptake buffer containing 1 μCi/ml [14C]methylammonium (MA) and various concentrations of unlabeled MA (20 μm–50 mm) with or without competitors or inhibitors. For some experiments, NaCl in SOS was replaced by KCl or N-methyl-d-glucamine. For acidification experiments, the internal oocyte pH (pHi) was altered using sodium acetate (14.Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 42: F516-F529Google Scholar, 15.Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar). Briefly, oocytes were preincubated for 25 min before uptake assay in buffers containing in mm 55 NaCl, 60 sodium acetate, 1.8 CaCl2, 1 MgCl2, 10 HEPES and adjusted to a final pH between 6.3 and 8.2. After 20–25 min, pHi achieves steady state, and recovery on removal requires 15 min (14.Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 42: F516-F529Google Scholar, 15.Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar). Fluxes were measured between 1 and 15 min. For all experiments, washing the oocytes seven times with 1.2 ml of ice-cold unlabeled uptake buffer stopped radiotracer uptake. Oocytes were solubilized in 200 μl of 5% SDS and analyzed for radioactivity in 5 ml of CytoSinct (ICN) by liquid scintillation counting. Water-injected control oocytes were evaluated in parallel in all assays, and control uptake values were subtracted from experimental values for cRNA-injected oocytes. Two-electrode voltage clamp was used to measure membrane currents in either cRNA-injected oocytes or water-injected control oocytes. Three days postinjection, single oocytes were perfused with solution containing in mm 96 NaCl, 0.2 CaCl2, 0.2 MgCl2, 10 HEPES, pH 7.5 by NaOH or with a solution containing 83 NaCl and 20 NH4Cl in place of 96 NaCl. Whole-oocyte membrane conductance was determined as described previously (16.Mak D.-O. D. McBride S. Raghuram V. Yun Y. Joseph S.K. Foskett J.K. J. Gen. Physiol. 2000; 115: 241-255Crossref PubMed Scopus (75) Google Scholar). Recombinant protein expression in oocytes was analyzed 3 days after cRNA injection by placing oocytes in 200 μl of oocyte extraction buffer in mm (150 NaCl, 100 Tris-HCl, 10 EDTA, pH 8.0) with protease inhibitors (1 mmphenylmethylsulfonyl fluoride, 1 μm pepstatin A, 1 μm leupeptin, 10 μm E-64) and homogenizing. The homogenate was placed on ice for 30 min and centrifuged for 5 min, and the supernatant was removed and centrifuged again at top speed for 30 min. The pellet was resuspended in sample loading buffer with 8m urea, heated at 37 °C for 15 min, analyzed by SDS-PAGE followed by immunoblotting with monoclonal antibody 2D10 (17.Mallinson G. Anstee D.J. Avent N.D. Ridgwell K. Tanner M.J. Daniels G.L. Tippett P. von dem Borne A.E. Transfusion. 1990; 30: 222-225Crossref PubMed Scopus (51) Google Scholar), and visualized with secondary horseradish peroxidase-conjugated anti-mouse IgG followed by ECL chemiluminescence (Amersham Biosciences). Experimental data were analyzed with Igor software (WaveMetrics, Lake Oswego, OR). Initial uptake rates were determined from slopes of linear fits to uptake measured over short periods (15 min) during which efflux was considered negligible. Apparent EC50 (K m) and V max values were derived by fitting experimental data with a simple Michaelis-Menten or Hill equation using a nonlinear least-squares fitting algorithm. IC50 of inhibition by ammonium was obtained by similar fitting of data with an inhibition Michaelis-Menton equation. Statistical significance was evaluated by two-tailed paired t test. A p value <0.05 was considered significant. Means are given as ±S.E. To test the hypothesis that Rh proteins are transporters or channels, we expressed Rh proteins in Xenopus oocytes by microinjection of cRNA for RhAG and/or RhCE and performed flux measurements on cRNA-injected and water-injected control oocytes. Expressed RhAG was detected in oocyte membrane-enriched fractions as a single band (Fig. 1 A), corresponding to the largest molecular weight band seen in native red blood cell membranes, suggesting that the expressed RhAG was fully glycosylated and properly trafficked to the plasma membrane. The detected protein was absent in water-injected oocytes. Expression of epitope-tagged RhCE was inconsistent and was only detected in membrane fractions when co-expressed with RhAG (data not shown). The characterization of ammonium ion transport is hindered by the lack of a convenient tracer for transport studies. Because13N label has a short half-life, is only measured by NMR, and is generally not available, we used the radioactive analog tracer MA, [14C]CH3NH3 +, which has been used in studies of ammonium transport in yeast and plants (18.Ninnemann O. Jauniaux J.-C. Frommer W.B. EMBO J. 1994; 13: 3464-3471Crossref PubMed Scopus (337) Google Scholar, 19.Kleiner D. Biochim. Biophys. Acta. 1981; 639: 41-52Crossref PubMed Scopus (235) Google Scholar, 20.Roon R.J. Even H.L. Dunlop P. Larimore F.L. J. Bacteriol. 1975; 122: 502-509Crossref PubMed Google Scholar). As Xenopus oocytes have an endogenous ammonium uptake system (21.Burckhardt M.-C. Fromter E. Pfluegers Arch. 1992; 420: 83-86Crossref PubMed Scopus (86) Google Scholar), water-injected control oocytes were evaluated in parallel. Expression of RhAG enhanced the rate of [14C]MA uptake compared with controls (Fig. 1 B). RhAG-mediated uptake was 8–10-fold greater at 15–30 min and was still 4–5-fold greater at 2 h compared with the linear rate of uptake seen in controls. RhCE cRNA-injected oocytes did not demonstrate uptake, and co-expression of RhCE with RhAG did not alter uptake (data not shown). To characterize the mechanisms of RhAG-induced MA uptake, we first examined the effects of modulating the membrane potential. When Na+ buffer (100 mm Na+, 1 mm K+) was replaced by high K+ buffer (1 mmNa+, 100 mm K+), the oocytes depolarized almost immediately, and the plasma membrane resting voltage went from an average of −35 to −9 mV. However, uptake of MA was unaffected (Fig. 2). This result suggested that uptake may not be a conductive process and furthermore that the transport mechanism is not coupled to the Na+gradient. To verify that uptake was independent of the Na+gradient, 100 mm Na+ in the uptake buffer was substituted with equimolar N-methyl-d-glucamine. RhAG-mediated uptake of MA was unaffected (data not shown). Two-electrode voltage clamp experiments were undertaken to determine the effects of RhAG expression on plasma membrane conductance properties. Perfusion with solutions containing 20 mmNH4Cl caused a small, albeit reproducible, increase in membrane conductance of ∼4–6 microsiemens greater than that observed in the water-injected controls (data not shown). The small enhancement of membrane conductance, taken together with the lack of effect of plasma membrane depolarization on MA uptake, suggests that RhAG-mediated MA uptake is not conductive. The rate of RhAG-mediated MA uptake was a saturable function of MA concentration, suggesting that the process was carrier-mediated. The data were well fitted with a Michaelis-Menten equation with the EC50 (K m) = 1.6 mm and V max = 190 pmol/oocyte/min (Fig. 3). We previously observed that weak complementation of the yeast ammonium transport deletion mutant by RhAG was pH-dependent. 2C. M. Westhoff, C. Burd, and J. K. Foskett, manuscript in preparation. MA uptake by oocytes in buffers with pH ranging from 5.5 to 8.5 revealed a significant increase in the RhAG-expressing oocytes at alkaline pH values (Fig. 4 A). Although elevated pH also enhanced MA uptake in control oocytes, the enhancement in RhAG-expressing oocytes was always at least 2-fold greater than in the controls. Uptake rates at pH 6.5 and below were low and not different from rates observed in the controls. Because the p K a of MA is 10.6, there was little change in the concentration of protonated MA over the range of pH values studied. The increased uptake rate observed at alkaline pH suggested that an outwardly directed proton gradient might be involved in MA uptake. To further clarify the observed pH dependence of MA uptake, the intracellular pH (pHi) of injected oocytes was manipulated. We reasoned that if an outwardly directed H+ ion gradient is involved in ammonium uptake, then acidification of the oocyte cytoplasm should enhance unidirectional RhAG-mediated MA uptake. A strategy that has been well documented for acidifying oocytes uses acetate-buffered solutions. In the presence of 60 mm sodium acetate, the lipophilic protonated form of acetic acid crosses the membrane and dissociates, acidifying intracellular pHi (14.Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 42: F516-F529Google Scholar, 15.Tsai T.D. Shuck M.E. Thompson D.P. Bienkowski M.J. Lee K.S. Am. J. Physiol. 1995; 268: C1173-C1178Crossref PubMed Google Scholar). After 20–25 min of incubation in acetate buffer the pHi reaches steady state and recovers on removal in ∼15 min (14.Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 42: F516-F529Google Scholar). Oocytes were preincubated for 25 min in acetate buffers adjusted from pH 6.1 to 7.4 and then moved to MA uptake buffer at pH 7.5 or 8.5, a treatment that is expected to lower pHi to approximately pH 5.9–6.8 (14.Choe H. Zhou H. Palmer L.G. Sackin H. Am. J. Physiol. 1997; 42: F516-F529Google Scholar). A decrease in pHi increased RhAG-specific MA uptake (Fig. 4 B), whereas control uptake rates were unaffected. In summary, RhAG-mediated MA uptake increased when either pHowas increased or when pHi was reduced. These results suggest that RhAG-specific MA uptake may be coupled to an outwardly directed proton gradient. Taken together with the lack of significant effect of RhAG expression on plasma membrane conductance and the lack of effect of changes in membrane potential on RhAG-mediated MA uptake, these data are consistent with uptake being mediated by MA-H+counter-transport. The Na+/H+ antiport inhibitor amiloride was without effect on uptake (Fig. 5 A). To characterize substrate specificity the effects of several amine-containing compounds were examined. RhAG-specific MA uptake was not affected by addition of the quaternary ammonium compounds tetramethyl- and tetraethylammonium chloride (Fig. 5 A), and neither urea nor glutamine inhibited MA uptake (Fig. 5 B). However, ammonium chloride was a strong competitor (Fig. 5 B). To compare relative affinities of NH4 and MA for the transport pathway, the inhibitor constant IC50 was determined by varying the concentration of NH4 and assessing the effects on RhAG-specific MA uptake (Fig. 5 C). The IC50 value for NH4 (1.1 mm) is close to the EC50 (1.6 mm) for MA uptake. This result suggests that NH4 may be the biologically relevant substrate for RhAG-mediated transport and that MA is an appropriate substrate analog for studying NH4 transport by Rh proteins. Although NH4Cl can acidify oocytes (21.Burckhardt M.-C. Fromter E. Pfluegers Arch. 1992; 420: 83-86Crossref PubMed Scopus (86) Google Scholar), which could affect the uptake of MA, the maximally inhibiting concentration of NH4 in our experiments was 2-fold lower than those normally used to acidify oocytes, and the half-maximal concentration was 1 mm, a concentration that is unlikely to produce significant pHi changes. However, a small intracellular acidification at these lower concentrations would result in an underestimation of the ability of NH4 to block MA uptake because acidification stimulates uptake. This report describes the first functional characterization of a human erythrocyte Rh protein. When expressed in Xenopusoocytes, RhAG was fully glycosylated, suggesting that it was processed appropriately and trafficked correctly to the oocyte plasma membrane. This result is important because erythrocyte Rh proteins have been notoriously difficult to express in heterologous cells. Indeed the recent expression of RhAG in the yeast ammonium transport mutant revealed that RhAG protein is not N-glycosylated and migrates at a lower molecular weight than that found in the native erythrocyte membrane (12.Marini A.M. Matassi G. Raynal V. Andre B. Cartron J.P. Cherif-Zahar B. Nat. Genet. 2000; 26: 341-344Crossref PubMed Scopus (303) Google Scholar). RhAG expression in those studies was shown to weakly complement the transport mutant, but it was not possible to demonstrate uptake of ammonium as the mechanism of complementation. We reasoned that the lack of glycosylation and weak complementation probably reflected inadequate processing and trafficking, which reduced the value of the yeast system for functional studies of Rh proteins.2 Oocytes expressing RhAG demonstrated an 8–10-fold increase in uptake of ammonium, measured with the radioactive analog [14C]MA, over water-injected controls. RhAG-mediated uptake of ammonium was unaffected by changes in the plasma membrane potential suggesting that the uptake pathway conferred by the expression of RhAG is not conductive. Uptake was independent of the plasma membrane Na+ gradient, but it was affected by a H+ gradient. Raising external pH or acidifying the oocyte cytoplasm increased the accumulation of the radioactive tracer. These results suggest some possible models for RhAG-mediated MA uptake and, by extension, for ammonium uptake. First, uptake may be coupled to proton extrusion. For mammalian cells, uptake of NH4 + would be predicted to acidify cells because the transported NH4 + can dissociate into NH3 and H+ in the cytoplasm. Uptake mediated by MA-H+ counter-transport would therefore be an attractive mechanism to accumulate ammonium without compromising pH. Second, our observed effects of pH on RhAG-mediated MA uptake might possibly be accounted for by changes in the concentration of the unprotonated NH3 molecule when pH is altered with the unprotonated molecule being the transported species. However, if the unprotonated molecule was the transported species, we would expect that the inhibition kinetics measured at pH 7.5 and pH 8.5 should be very different since the p K a of ammonium (9.25) dictates that the unprotonated form (NH3) is less than 2% of the total ammonium concentration at pH 7.5 but increases to ∼15% at pH 8.5. In contrast, the same IC50 (1.1 mm) was measured at both pH values, arguing that the transported species is not the unprotonated molecule. Third, the effects of pH on the observed MA uptake rates could possibly be the result of allosteric effects of H+. However, this possibility seems unlikely since the pH gradient, rather than the pH on either side of the membrane, appears to be most important. We conclude therefore that the most reasonable model that can account for our results is one in which RhAG-specific MA uptake is mediated by an exchange of cationic MA (and by extension NH4 +) with H+. Further studies are underway to conclusively determine whether RhAG is an antiporter. Transport across membranes is required to efficiently scavenge NH4 + for use as a nitrogen source in bacteria, yeast, and higher plants; however, little is known about the requirements for NH4 + transport in mammals. It was long assumed that high passive membrane permeability of ammonia (NH3) would obviate the need for specific transport pathways in mammalian cells (19.Kleiner D. Biochim. Biophys. Acta. 1981; 639: 41-52Crossref PubMed Scopus (235) Google Scholar). Water and urea permeation were originally thought to occur by passive movement through the lipid bilayer but are now known to be mediated by specific proteins. Similarly, rates of passive diffusion are not sufficient to account for the movement of NH4 + in a number of organs (22.Nagaraja T.N. Brookes N. Am. J. Physiol. 1998; 274: C883-C891Crossref PubMed Google Scholar, 23.Singh S.K. Binder H.J. Geibel J.P. Boron W.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11573-11577Crossref PubMed Scopus (101) Google Scholar, 24.Knepper M.A. Packer R. Good D.W. Physiol. Rev. 1989; 69: 179-249Crossref PubMed Scopus (241) Google Scholar). NH4 + movement in the kidney tubular segments is transport-mediated. Uptake is thought to be due, in part, to the ability of NH4 + to substitute for K+ on the Na+-K+-2Cl−co-transporter (25.Kinne R. Kinne-Saffran E. Shutz H. Scholermann B. J. Membr. Biol. 1986; 94: 279-284Crossref PubMed Scopus (115) Google Scholar) and the Na+,K+-ATPase (26.Wall S.M. Miner. Electrolyte Metab. 1996; 22: 311-317PubMed Google Scholar) or to be carried by the Na+/H+ exchanger (27.Kinsella J.L. Aronson P.S. Am. J. Physiol. 1981; 241: C220-C226Crossref PubMed Google Scholar). Our results suggest that ammonium transport may also be mediated specifically by Rh proteins. The involvement of Rh proteins in membrane transport has been suspected recently based on predicted topology and weak similarity to ammonium transporters. However, until now direct demonstration of the movement of a specific substrate by Rh proteins has not been shown. Here we show that ammonium is a biologically relevant substrate for RhAG-mediated transport and that MA is an appropriate substrate analog for studying transport by the growing family of Rh-related proteins. Although ascribing a role for NH4 + transport by erythrocytes is somewhat speculative at present, the large total erythrocyte mass may enable these cells to carry a significant amount of ammonium or ammonia possibly to be exchanged in the liver and kidney where other Rh-related proteins are expressed. Indeed physiologic ammonium concentrations in blood plasma are low, 0.1–0.2 mm, but erythrocyte ammonia levels average 3 times greater (28.Huizenga J.R. Tangerman A. Gips C.H. Ann. Clin. Biochem. 1994; 31: 529-543Crossref PubMed Scopus (83) Google Scholar). The erythrocyte RhAG-mediated membrane transport of NH4 + might have implications in metabolic disorders, especially those associated with hyperammonemia. We thank A. E. von dem Borne, Netherlands Red Cross, Amsterdam for monoclonal antibody 2D10 and Don Siegel for access to laboratory space and equipment.