AQP1 Promoter Variant, Water Transport, and Outcomes in Peritoneal Dialysis.

BACKGROUND: Variability in ultrafiltration influences prescriptions and outcomes in patients with kidney failure who are treated with peritoneal dialysis. Variants in AQP1, the gene that encodes the archetypal water channel aquaporin-1, may contribute to that variability. METHODS: We gathered clinical and genetic data from 1851 patients treated with peritoneal dialysis in seven cohorts to determine whether AQP1 variants were associated with peritoneal ultrafiltration and with a risk of the composite of death or technique failure (i.e., transfer to hemodialysis). We performed studies in cells, mouse models, and samples obtained from humans to characterize an AQP1 variant and investigate mitigation strategies. RESULTS: The common AQP1 promoter variant rs2075574 was associated with peritoneal ultrafiltration. Carriers of the TT genotype at rs2075574 (10 to 16% of patients) had a lower mean (±SD) net ultrafiltration level than carriers of the CC genotype (35 to 47% of patients), both in the discovery phase (506±237 ml vs. 626±283 ml, P = 0.007) and in the validation phase (368±603 ml vs. 563±641 ml, P = 0.003). After a mean follow-up of 944 days, 139 of 898 patients (15%) had died and 280 (31%) had been transferred to hemodialysis. TT carriers had a higher risk of the composite of death or technique failure than CC carriers (adjusted hazard ratio, 1.70; 95% confidence interval [CI], 1.24 to 2.33; P = 0.001), as well as a higher risk of death from any cause (24% vs. 15%, P = 0.03). In mechanistic studies, the rs2075574 risk variant was associated with decreases in AQP1 promoter activity, aquaporin-1 expression, and glucose-driven osmotic water transport. The use of a colloid osmotic agent mitigated the effects of the risk variant. CONCLUSIONS: A common variant in AQP1 was associated with decreased ultrafiltration and an increased risk of death or technique failure among patients treated with peritoneal dialysis. (Funded by the Swiss National Science Foundation and others.).

Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1).

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3- and Cl- exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl-/HCO3- exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3- and outward Cl- gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3- for stomatin-deficient patients. Similarly, kinetics of Cl- efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

Evidence of a structural and functional ammonium transporter RhBG·anion exchanger 1·ankyrin-G complex in kidney epithelial cells.

The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.

Energetic and molecular water permeation mechanisms of the human red blood cell urea transporter B.

Urea transporter B (UT-B) is a passive membrane channel that facilitates highly efficient permeation of urea. In red blood cells (RBC), while the major function of UT-B is to transport urea, it is assumed that this protein is able to conduct water. Here, we have revisited this last issue by studying RBCs and ghosts from human variants with defects of aquaporin 1 (AQP1) or UT-B. We found that UT-B's osmotic water unit permeability (pfunit) is similar to that of AQP1. The determination of diffusional permeability coefficient (Pd) allowed the calculation of the Pf/Pd ratio, which is consistent with a single-file water transport. Molecular dynamic simulations of water conduction through human UT-B confirmed the experimental finding. From these results, we propose an atomistic description of water-protein interactions involved in this permeation. Inside the UT-B pore, five water molecules were found to form a single-file and move rapidly along a channel by hydrogen bond exchange involving two critical threonines. We further show that the energy barrier for water located in the central region coincides with a water dipole reorientation, which can be related to the proton exclusion observed experimentally. In conclusion, our results indicate that UT-B should be considered as a new member of the water channel family.

Rapid Cl⁻/HCO⁻3exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells.

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

In vitro generated Rh(null) red cells recapitulate the in vivo deficiency: a model for rare blood group phenotypes and erythroid membrane disorders.

Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells.

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH(3) transport through the channel and NH(3) diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh(null) lacking RhAG, we observed that alkalinization rate constant values decreased ∼50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.

A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia.

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in bone marrow. We have identified a missense mutation in KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.

A functional AQP1 allele producing a Co(a-b-) phenotype revises and extends the Colton blood group system.

BACKGROUND: The Colton blood group system currently comprises three antigens, Co(a) , Co(b) , and Co3. The latter is only absent in the extremely rare individuals of the Colton "null" phenotype, usually referred to as Co(a-b-), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a-b-) individual with no AQP1 deficiency suggested another molecular basis for the Co(a-b-) phenotype. STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped-flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction-restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells. RESULTS: Although Co(a-b-), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single-amino-acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K-562 cells, the Q47R substitution was shown to inhibit the expression of both Co(a) and Co(b) antigens. Other naturally occurring single-amino-acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system. CONCLUSIONS: The Co(a-b-) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Co(b) antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.

Functional reconstitution into liposomes of purified human RhCG ammonia channel.

BACKGROUND: Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3), human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. METHODOLOGY/PRINCIPAL FINDINGS: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12)E(8) detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH(3) transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. CONCLUSIONS/SIGNIFICANCE: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3) (around 1x10(-3) microm(3)xs(-1)) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10(-3) microm(3)xs(-1)), and in human red blood cells endogenously expressing RhAG (2.18x10(-3) microm(3)xs(-1)). The major finding of this study is that RhCG protein is active as an NH(3) channel and that this function does not require any protein partner.

Generation and characterisation of Rhd and Rhag null mice.

Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.

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.

Phosphorylation and ankyrin-G binding of the C-terminal domain regulate targeting and function of the ammonium transporter RhBG.

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH(4)(+) deprotonation takes place at S1 before NH(3) conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

RhAG protein of the Rhesus complex is a CO2 channel in the human red cell membrane.

We have determined CO2 permeabilities, P(CO2), of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While P(CO2) of normal red cells is approximately 0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rh(null)) a significantly reduced P(CO2) of 0.07 cm/s +/-0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1(null)), suggesting that each of the Rh and AQP-1 proteins is responsible for approximately 1/2 of the normal CO2 permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO2 permeability. The CO2 pathway constituted by Rh proteins was inhibitable at pH(e)= 7.4 by NH4Cl with an I50 of approximately 10 mM corresponding to an I50 for NH3 of approximately 0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH4Cl/NH3. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal P(CO2), while in contrast another AE1 inhibitor, DiBAC, does not inhibit P(CO2), although it markedly reduces P(HCO3-). We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO2 in addition to NH3.

Reprint of "Structural and mechanistic aspects of Amt/Rh proteins" [J. Struct. Biol. 158 (2007) 472-481].

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport. The Escherichia coli channel AmtB has become the model system of choice for analysis of the mechanism of ammonia conductance, increasingly also through molecular dynamics simulations. Further progress in a more detailed mechanistic understanding of these proteins requires a reliable in vitro assay using purified protein, allowing quantitative kinetic measurements under a variety of experimental conditions for different Amt/Rh proteins, including mutants. Here, we critically review the existing functional data in the context of the most interesting and unresolved mechanistic questions and we present our results, obtained using an in vitro assay set up with the purified E. coli channel AmtB.

Structural and mechanistic aspects of Amt/Rh proteins.

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport. The Escherichia coli channel AmtB has become the model system of choice for analysis of the mechanism of ammonia conductance, increasingly also through molecular dynamics simulations. Further progress in a more detailed mechanistic understanding of these proteins requires a reliable in vitro assay using purified protein, allowing quantitative kinetic measurements under a variety of experimental conditions for different Amt/Rh proteins, including mutants. Here, we critically review the existing functional data in the context of the most interesting and unresolved mechanistic questions and we present our results, obtained using an in vitro assay set up with the purified E. coli channel AmtB.

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

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.

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

Rhesus (Rh) antigens are carried by a membrane complex that includes Rh proteins (D and CcEe), Rh-associated glycoproteins (RhAG), and accessory chains (LW and CD47) associated by noncovalent bonds. In heterologous expression systems, RhAG and its kidney orthologs function as ammonium transporters. In red blood cells (RBCs), it is generally accepted that NH(3) permeates by membrane lipid diffusion. We have revisited these issues by studying RBC and ghosts from human and mouse genetic variants with defects of proteins that comprise the Rh complex. In both normal and mutant cells, stopped-flow analyses of intracellular pH changes in the presence of inwardly directed methylammonium (CH(3)NH(+)(3)+CH(3)NH(2)) or ammonium (NH(+)(4)+NH(3)) gradients showed a rapid alkalinization phase. Cells from human and mouse variants exhibited a decrease in their kinetic rate constants that was strictly correlated to the degree of reduction of their RhAG/Rhag expression level. Rate constants were not affected by a reduction of Rh, CD47, or LW. CH(3)NH(2)/NH(3) transport was characterized by (i) a sensitivity to mercurials that is reversible by 2-mercaptoethanol and (ii) a reduction of alkalinization rate constants after bromelain digestion, which cleaves RhAG. The results show that RhAG facilitates CH(3)NH(2)/NH(3) movement across the RBC membrane and represents a potential example of a gas channel in mammalian cells. In RBCs, RhAG may transport NH(3) to detoxifying organs, like kidney and liver, and together with nonerythroid tissue orthologs may contribute to the regulation of the systemic acid-base balance.

Characterization of urea transport in Bufo arenarum oocytes.

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.