Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis.

We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.     

Structural involvement in substrate recognition of an essential aspartate residue conserved in Mep/Amt and Rh-type ammonium transporters

Ammonium transport proteins belonging to the Mep/Amt/Rh family are spread throughout all domains of life. A conserved aspartate residue plays a key role in the function of Escherichia coli AmtB. Here, we show that the analogous aspartate residue is critical for the transport function of eukaryotic family members as distant as the yeast transporter/sensor Mep2 and the human RhAG and RhCG proteins. In yeast Mep2, replacement of aspartate¹⁸⁶ with asparagine produced an inactive transporter localized at the cell surface, whilst replacement with alanine was accompanied by stacking of the protein in the endoplasmic reticulum. Introduction of an acidic residue, glutamate, produced a partially active protein. A carboxyl group at position 186 of Mep2 therefore appears mandatory for function. Kinetic analysis shows the Mep2^D186E variant to be particularly affected at the level of substrate affinity, suggesting an involvement of aspartate¹⁸⁶ in ammonium recognition. Our data also put forward that ammonium recognition and/or transport by Mep2 is required for the sensor role played in the development of pseudohyphal growth. Finally, replacement of the conserved aspartate with asparagine in human RhAG and RhCG proteins resulted in the loss of bi-directional transport function. Hence, this aspartate residue might play a preserved functional role in Mep/Amt/Rh proteins.     

Structural and functional insights into the AmtB/Mep/Rh protein family.

X-ray crystallography revealed a similar architecture of the ammonium transport protein AmtB from Escherichia coli and the homologous protein Amt-1 from Archaeoglobus fulgidus. Furthermore, the atomic structures suggest that the proteins conduct ammonia (NH3) rather than ammonium ions (NH4+). These findings indicate that the more than 350 members of the ammonium transporter/methylamine permease/Rhesus (Amt/Mep/Rh) protein family found in archaea, bacteria, fungi, plants and animals are ammonia-conducting channels rather than ammonium ion transporters. The essential part of these proteins is the narrow hydrophobic ammonia-conducting pore with two highly conserved histidine residues located in the middle of the pore. A specific ammonium ion binding site is found at the extracellular entry site of E. coli AmtB. E. coli AmtB and its regulator GlnK form an effective ammonium sensory system that couples intracellular gene regulation by the nitrogen control system to external changes in ammonium availability. Based on structural and functional analysis of various mutants, two conserved histidine residues were found to be essential for substrate conductance also in the functional eukaryotic ammonium transporters. The next big challenge in the field surely is to determine the atomic structure of Rh proteins.     

From yeast ammonium transporters to Rhesus proteins, isolation and functional characterization.

Saccharomyces cerevisiae possesses three ammonium transporters from the Mep/Amt family involved in ammonium acquisition and retention. We have shown that Rh proteins are structurally related to Mep/Amt proteins and that human RhAG and RhCG perform bi-directional ammonium transport upon heterologous expression in yeast. Using yeast as an expression tool, we have started a structure-function analysis of distinct members from the Mep/Amt/Rh super-family.     

Amt/MEP/Rh proteins conduct ammonia

The structure determination of the ammonium transport protein AmtB from Escherichia coli strongly indicates that the members of the ubiquitous ammonium transporter/methylamine permease/Rhesus (Amt/MEP/Rh) protein family are ammonia-conducting channels rather than ammonium ion transporters. The most conserved part of these proteins, apart from the common overall structure with 11 transmembrane helices, is the pore lined by hydrophobic side chains except for two highly conserved histidine residues. A high-affinity ion-binding site specific for ammonium is present at the extracellular pore entry of the Amt/MEP proteins. It is proposed to play an important role in enhancing net transport at very low external ammonium concentrations and to provide discrimination against water. The site is not conserved in the animal Rhesus proteins which are implicated in ammonium homeostasis and saturate at millimolar ammonium concentrations. Many aspects of the biological function of these ammonia channels are still poorly understood and further studies in cellular systems are needed. Likewise, studies with purified, reconstituted Amt/MEP/Rh proteins will be needed to resolve open mechanistic questions and gain a more quantitative understanding of the conduction mechanism in general and for different subfamily representatives.     

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins.

The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.     

Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels.

Physiological evidence from our laboratory indicates that Amt/Mep proteins are gas channels for NH3, the first biological gas channels to be described. This view has now been confirmed by structural evidence and is displacing the previous belief that Amt/Mep proteins were active transporters for the NH4+ ion. Still disputed is the physiological substrate for Rh proteins, the only known homologues of Amt/Mep proteins. Many think they are mammalian ammonium (NH4+ or NH3) transporters. Following Monod's famous dictum, "Anything found to be true of E. coli must also be true of elephants" [Perspect. Biol. Med. 47(1) (2004) 47], we explored the substrate for Rh proteins in the unicellular green alga Chlamydomonas reinhardtii. C. reinhardtii is one of the simplest organisms to have Rh proteins and it also has Amt proteins. Physiological studies in this microbe indicate that the substrate for Rh proteins is CO2 and confirm that the substrate for Amt proteins is NH3. Both are readily hydrated gases. Knowing that transport of CO2 is the ancestral function of Rh proteins supports the inference from hematological research that a newly evolving role of the human Rh30 proteins, RhCcEe and RhD, is to help maintain the flexible, flattened shape of the red cell.     

Transport mechanisms in the ammonium transporter family

Ammonium transport is mediated by membrane proteins of the ubiquitous Amt/Rh family. Despite the availability of different X-ray structures that provide many insights on the ammonium permeation process, the molecular details of its mechanism remain controversial. The X-ray structures have revealed that the pore of the Amt and Rh proteins is characterized by a hydrophobic portion about 12 Å long in which electronic density was observed in crystallographic study of AmtB from Escherichia coli. This electronic density was initially only observed when crystals were grown in presence of ammonium salt and was thus attributed to ammonia (NH₃) molecules, and lead the authors to suggest that the conduction mechanism in the Amt/Rh proteins involves the single-file diffusion of NH₃ molecules. However, other X-ray crystallography results and molecular mechanics simulations suggest that the pore of AmtB could also be filled with water molecules. The possible presence of water molecules in the pore lumen calls for a reassessment of the growing consensus that Amt/Rh proteins work as plain NH₃ channels. Indeed, functional experiments on plant ammonium transporters and rhesus proteins suggest a variety of permeation mechanisms including the passive diffusion of NH₃, the antiport of NH₄⁺/H⁺, the transport of NH₄⁺, or the cotransport of NH₃/H⁺. We discuss these mechanisms in light of some recent functional and simulation studies on the AmtB transporter and illustrate how they can be reconciled with the available high resolution X-ray data.     

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.     

Rh proteins vs Amt proteins: an organismal and phylogenetic perspective on CO2 and NH3 gas channels.

Rh (Rhesus) proteins are homologues of ammonium transport (Amt) proteins. Physiological and structural evidence shows that Amt proteins are gas channels for NH(3), but the substrate of Rh proteins, be it CO2 as shown in green alga, or NH3/NH4+ as shown in mammalian cells, remains disputed. We assembled a large dataset generated of Rh and Amt to explore how Rh originated from and evolved independently of Amt relatives. Analysis of this rich data implies that Rh was split from Amt first to emerge in archaeal species. The Rh ancestor underwent divergence and duplication along speciation, leading to neofunctionalization and subfunctionalization of the Rh family. The characteristic organismal distribution of Rh vs. Amt reflects their early separation and subsequent independent evolution: they coexist in microbes and invertebrates but do not in fungi, vascular plants or vertebrates. Rh gene-duplication was prominent in vertebrates: while epithelial RhBG/RhCG displayed strong purifying selection, erythroid Rh30 and RhAG experienced different episodes of positive selection in each of which adaptive evolution occurred at certain time points and in a few codon sites. Mammalian Rh30 and RhAG were subject to particularly strong positive selection in some codon sites in the lineage from rodents to human. The grounds of this adaptive evolution may be driven by the necessity to increase the surface/volume ratio of biconcave erythrocytes for facilitative gas diffusion. Altogether, these results are consistent with Rh proteins not being the orthologue of Amt proteins but having gained the function for CO2/HCO3- transport, with important roles in systemic pH regulation.     

Ammonium ion transport by the AMT/Rh homolog TaAMT1;1 is stimulated by acidic pH

It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH ₄ ⁺ or methylammonium ions (MeA⁺). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH ₄ ⁺ or MeA⁺ transport. This was confirmed by the close correlation between the uptake of radio-labeled MeA⁺ and MeA⁺-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH ₄ ⁺ and MeA⁺. We suggest a model where NH ₄ ⁺ is transported as NH₃ and H⁺ via separate pathways but the latter two recombine before leaving the protein.     

Expression of the non-erythroid Rh glycoproteins in mammalian tissues.

A novel family of proteins, the Mep/AMT/Rh glycoprotein family may mediate important roles in transmembrane ammonia transport in a wide variety of single-celled and multicellular organisms. Results from our laboratory have examined the expression of the non-erythroid proteins, Rh B Glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in a wide variety of mammalian tissues. In the kidney, Rhbg and Rhcg are present in distal nephron sites responsible for ammonia secretion. In the mouse kidney, Rhbg immunoreactivity is exclusively basolateral and Rhcg immunoreactivity is exclusively apical, whereas in the rat kidney Rhcg exhibits both apical and basolateral expression. Chronic metabolic acidosis increases Rhcg expression in the outer and inner medulla of the rat kidney; these changes, at least in the outer medullary collecting duct, involve changes in total cellular protein expression in both principal and intercalated cell and changes in its subcellular localization. In the liver, Rhbg is present in the basolateral plasma membrane of the perivenous hepatocyte and Rhcg is present in bile duct epithelia. In the gastrointestinal tract, Rhbg and Rhcg exhibit cell-specific, axially heterogeneous, and polarized expression. These patterns of expression are consistent with Rhbg and Rhcg mediating important roles in mammalian ammonia biology. The lack of the effect of chronic metabolic acidosis on Rhbg expression raises the possibility that Rhbg may function either as ammonia sensing-protein or that it may mediate roles other than ammonia transport.     

Physiological role of the putative ammonium transporter RhCG in the mouse.

Ammonium excretion into urine is a major process essential to the regulation of acid-base homeostasis. We have shown that Rh-type proteins, including renal RhCG, belong to the Mep/Amt family of ammonium transporters and promote bi-directional ammonium transport upon heterologous expression in yeast. To study the physiological role of RhCG and to test a potential function in ammonium excretion, we have generated mice bearing an invalidation of the corresponding gene.     

Rh glycoproteins in epithelial cells: lessons from rat and mice studies.

Rhesus glycoproteins are a recently discovered family of ammonium transporters and a new branch of the Mep/AMT proteins superfamily that was identified more than 15 years ago in lower organisms and plants. Despite many ex vivo studies showing evidences that Rh glycoproteins can accelerate transmembrane NH3 or NH4+ transfer, their role in normal and disease physiology remains unknown. This review focuses on some of the different studies carried out in animal models to gain insight into Rh glycoprotein function. Immunolocalization studies have added new evidence that this protein family is related to ammonium transport or metabolism in epithelial cells. However, the absence of distal tubular acidosis or hyperammonemia in Rhbg KO mice have raised new questions about the physiological significance of these proteins.     

Non-erythroid Rh glycoproteins: a putative new family of mammalian ammonium transporters

The Rhesus (Rh) glycoproteins, originally described in human blood cells, are mostly recognized for their immunogenic characteristics and importance in pregnancy. The Rh proteins in the red blood cell are expressed as an "Rh complex" made up of one D-subunit, one CE-subunit and two Rh-associated glycoprotein (RhAG) subunits. In addition to its antigenic property, the Rh complex is thought to contribute to membrane stability and structure of red blood cells. The exact function is yet to be determined. Recently, two non-erythroid Rh glycoproteins were cloned from mice (Rhcg and Rhbg) and humans (RhCG and RhBG). RhCG is expressed at the membrane surface alone with no apparent need for heteromeric interaction with other glycoproteins. It is more similar to RhAG than to Rh CE/D, occurs late in development and is expressed abundantly and broadly in kidney and testis. In the kidney RhCG is localized to the apical cell membrane of the collecting duct. Rhbg and its human analog (RhBG) are expressed mainly in liver, skin and the kidney tubules. In the kidney collecting duct, Rhbg is localized to the basolateral membrane. Based on structural similarities to the methylammonium and ammonium permease/ammonium (MEP/Amt) transporters in yeast and their sequence homology, these proteins probably function as NH(4)(+) transporters. An initial study has indicated that RhAG or RhCG promote efflux of NH(4)(+), whereas another study has suggested that RhAG functions as an NH(4)(+)-H(+) exchanger. Evidence for such a function is still circumstantial and data indicating that Rh proteins function as NH(4)(+) transporters are indirect.     

Influenza virus M2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds

The oligomeric structure of the influenza A virus M2 integral membrane protein was determined. On SDS-polyacrylamide gels under nonreducing conditions, the influenza A/Udorn/72 virus M2 forms disulfide-linked dimers (30 kDa) and tetramers (60 kDa). Sucrose gradient analysis and chemical cross-linking analysis indicated that the oligomeric form of M2 is a tetramer consisting of either a pair of disulfide-linked dimers or disulfide-linked tetramers. In addition, a small amount of a cross-linked species of 150-180,000 kDa, which the available data suggest contains only M2 polypeptides, was observed. The role of M2 cysteine residues in disulfide bond formation and their role in forming oligomers were examined by converting each of the two extracellular and single cytoplasmic cysteine residues to serine residues and expressing the altered M2 proteins in eukaryotic cells. Removal of either one of the N-terminal cysteines at residues 17 or 19 indicated that tetramers formed that consisted of a pair of noncovalently associated disulfide-linked dimers, suggesting that each of the cysteine residues is equally competent for forming disulfide bonds. When both cysteine residues were removed from the M2 N-terminal domain, no disulfide-linked forms were observed. When solubilized in detergent this double-cysteine mutant lost reactivity with a M2-specific mAb and exhibited an altered sedimentation pattern on sucrose gradients. However, chemical cross-linking of this double-cysteine mutant in membranes indicated that it can form tetramers. Taken together, these data suggest that disulfide bond formation, although not essential for oligomeric assembly, stabilizes the M2 tetramer from disruption by detergent solubilization.     

Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501

The nitrogen-fixing Pseudomonas stutzeri strain A1501 contains two ammonium transporter genes, amtB1 and amtB2, linked to glnK. Growth of an amtB1-amtB2 double deletion mutant strain was not impaired compared to that of the wild type under any conditions tested, and it was still capable of taking up ammonium ions at nearly wild-type rates. Nitrogenase activity was repressed in wild-type strain A1501 in response to the addition of ammonium, but nitrogenase activity was only partially impaired in the amtB1 and amtB2 double mutant, suggesting that the two AmtB proteins are involved in regulating expression of nitrogenase or its activity in response to ammonium. An interaction between GlnK and AmtB1 or AmtB2 was observed in a yeast two-hybrid assay. Ammonium was excreted by the amtB double mutant strain under nitrogen fixation conditions, particularly when nifA was expressed constitutively. This suggests that AmtB proteins play a role in controlling the internal pool of ammonia within the cell.     

Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.

We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.     

The role of Lys2–Cl−–Lys2 salt linkages in oligomeric intermediates of RbsD protein in Escherichia coli

As a homo-oligomeric protein, the disassembly of Escherichia coli RbsD decamer produces a urea-unfolded oligomeric intermediate structure, as the dissociation speed of the protein is lower than that of the unfolding process. There are five Lys2–Cl⁻–Lys2 salt linkages to connect these subunits. To explore the role of the salt linkages in these oligomeric intermediates, the Lys2Ala mutated in the N-terminal of E. coli RbsD protein subunit was designed. It was found that the RbsD mutation protein (RbsD:K2A) loses its minor larger oligomers, which exist in RbsD, and displays other several oligomeric states (less than decamers), meanwhile the state of the oligomers depends on the protein concentration. It was also found that compared with RbsD, the crosslinking capability of the subunits of RbsD:K2A is weaker, while the crosslinking rate of dimers is higher, RbsD:K2A needs to substantially adjust its conformation to meet the space requirements when combined with d -ribose. On the basis of these results, we suggest that Lys2–Cl⁻–Lys2 salt linkages in E. coli RbsD protein play an important role in stabilizing the intermediate products of oligomers and maintaining interaction between the intermediate products of oligomers, which may shed light on the study of these oligomeric proteins.