Structural Features of the Glutamate Transporter Family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C₄-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C₄-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning α-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Structural features of the glutamate transporter family.

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.     

Glutamate transporters combine transporter- and channel-like features

Glutamate transporters in the mammalian central nervous system have a unique position among secondary transport proteins as they exhibit glutamate-gated chloride-channel activity in addition to glutamate-transport activity. In this article, the available data on the structure of the glutamate transporters are compared with high-resolution crystal structures of channel proteins. In addition, binding-site properties of glutamate transporters, and the ligand-binding site of an ionotropic glutamate receptor of which the crystal structure is known, are compared. Possible structural solutions for the combination of channel and transporter activity in one membrane protein are proposed.     

Sequence and structure of the yeast galactose transporter.

The previously cloned GAL2 gene of the Saccharomyces cerevisiae galactose transporter has been sequenced. The nucleotide sequence predicts a protein with 574 amino acids (Mr, 63,789). Hydropathy plots suggest that there are 12 membrane-spanning segments. The galactose transporter shows both sequence and structural homology with a superfamily of sugar transporters which includes the human HepG2-erythrocyte and fetal muscle glucose transporters, the rat brain and liver glucose transporters, the Escherichia coli xylose and arabinose permeases, and the S. cerevisiae glucose, maltose, and galactose transporters. Sequence and structural motifs at the N-terminal and C-terminal regions of the proteins support the view that the genes of this superfamily arose by duplication of a common ancestral gene. In addition to the sequence homology and the presence of the 12 membrane-spanning segments, the members of the superfamily show characteristic lengths and distributions of the charged, hydrophilic connecting loops. There is indirect evidence that the transporter is an N-glycoprotein. However, its only N-glycosylation site occurs in a charged, hydrophilic segment. This could mean that this segment is part of a hydrophilic channel in the membrane. The transporter has a substrate site for the cyclic AMP-dependent protein kinase which may be a target of catabolite inactivation. The transporter lacks a strong sequence enriched for proline (P), glutamate (E), aspartate, serine (S), and threonine (T) and flanked by basic amino acids (PEST sequence) even though it has a short half-life. Mechanisms for converting the poor PEST to a possible PEST sequence are considered. Like the other members of the superfamily, the galactose transporter lacks a signal sequence.     

Structural and mechanistic diversity of secondary transporters

Recent reports on the three-dimensional structure of secondary transporters have dramatically increased our knowledge of the translocation mechanism of ions and solutes. The structures of five transporters at atomic resolution have yielded four different folds and as many different translocation mechanisms. The structure of the glutamate transporter homologue GltPh confirmed the role of pore-loop structures as essential parts of the translocation mechanism in one family of secondary transporters. Biochemical evidence for pore-loop structures in several other families suggest that they might be common in secondary transporters, adding to the structural and mechanistic diversity of secondary transporters.     

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.     

Membrane topology of the Mep/Amt family of ammonium transporters

The Mep/Amt proteins constitute a new family of transport proteins that are ubiquitous in nature. Members from bacteria, yeast and plants have been identified experimentally as high-affinity ammonium transporters. We have determined the topology of AmtB, a Mep/Amt protein from Escherichia coli, as a representative protein for the complete family. This was established using a minimal set of AmtB-PhoA fusion proteins with a complementary set of AmtB-LacZ fusions. These data, accompanied by an in silico analysis, indicate that the majority of the Mep/Amt proteins contain 11 membrane-spanning helices, with the N-terminus on the exterior face of the membrane and the C-terminus on the interior. A small subset, including E. coli AmtB, probably have an additional twelfth membrane-spanning region at the N-terminus. Addition of PhoA or LacZ alpha-peptide to the C-terminus of E. coli AmtB resulted in complete loss of transport activity, as judged by measurements of [14C]-methylammonium uptake. This C-terminal region, along with four membrane-spanning helices, contains multiple residues that are conserved within the Mep/Amt protein family. Structural modelling of the E. coli AmtB protein suggests a number of secondary structural features that might contribute to function, including a putative ammonium binding site on the periplasmic face of the membrane at residue Asp-182. The implications of these results are discussed in relation to the structure and function of the related human Rhesus proteins.     

Mutating a Conserved Proline Residue within the Trimerization Domain Modifies Na+ Binding to Excitatory Amino Acid Transporters and Associated Conformational Changes

Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.     

Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury

Glutamate excitotoxicity plays an important role in the development of secondary injuries that occur following traumatic brain injury (TBI), and contributes significantly to expansion of the total volume of injury. Acute increases in extracellular glutamate levels have been detected in both experimental brain trauma models and in human patients, and can lead to over-stimulation of glutamate receptors, resulting in a cascade of excitotoxic-related mechanisms culminating in neuronal damage. These elevated levels of glutamate can be effectively controlled by the astrocytic glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2). However, evidence indicate these transporters and splice variant are downregulated shortly following the insult, which then precipitates glutamate-mediated excitotoxic conditions. Lack of success with glutamate receptor antagonists as a potential source of clinical intervention treatment following TBI has resulted in the necessity for a better understanding of the mechanisms that underlie the process of excitotoxicity, including the function and regulation of glutamate transporters. Such new insight should improve the likelihood of development of novel avenues for therapeutic intervention following TBI.     

高亲和力谷氨酸转运体结构和功能的研究进展

真核生物高亲和力谷氨酸转运体(excitatory amino acid transporters,EAATs)分为GLAST(EAAT1)、GLT-1(EAAT2)、EAAC1(EAAT3)、EAAT4和EAAT5等5个亚型.高亲和力谷氨酸转运体结构学的研究,揭示了谷氨酸转运体的跨膜拓扑结构、真核和原核生物EAATs结构的差异,以及在底物转运过程中的一些底物和协同转运离子的结合位点.其功能学的研究发现,EAATs在参与突触的传递,避免兴奋性氨基酸的毒性效应中发挥重要作用,同时也参与了对学习、记忆以及运动行为的调控.结合我们既往的工作,就近几年EAATs的结构和功能研究做一综述.     

Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT

The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404. Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity. Cysteine mutations at the other positions were well tolerated. Only the two most amino- and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop structure outside the membrane. The labeling pattern of these mutants using membrane- permeable and -impermeable thiol reagents showed that the N and C termini of the mutated stretch are located extra- and intracellularly, respectively. Thus, the location of the membrane-spanning segment was confined to a stretch of 23 residues between Gly-374 and Ser-399. Cysteine residues in three mutants in the central part of the segment (M381C, V388C, and N391C) could be labeled with the small and flexible reagent 2-aminoethyl methanethiosulfonate hydrobromide only, suggesting accessibility via a narrow aqueous pore. When the region was modeled as an alpha-helix, all positions at which cysteine mutations lead to inactive or severely impaired transporters cluster on one face of this helix. The inactive mutants showed neither proton motive force-driven uptake activity nor exchange activity nor glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic alpha-helix. The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.     

Loss of cell viability by histidine substitution of leucine 325 of the glutamate transporter EAAT1

Although glutamate transporters and neutral amino acid transporters have 55% amino acid identity in the transmembrane domains, many residues are still unique to individual transporters, providing for structural stability or substrate binding. In this study, the mutant protein L325H, which replaced a leucine 325 of the glutamate transporter EAAT1 by a histidine, was evaluated. When expressed in Xenopus oocytes, L325H caused oocytes to weaken pigmentation in the animal pole, accompanied by patches of colorless spots. Oocytes finally oozed cytoplasm. The resting membrane potential in L325H oocytes was -18.9 +/- 2.5 mV, significantly more positive than -37.3 +/- 2.5 mV of oocytes expressing EAAT1. The holding current at -60 mV was 283.1 +/- 48.3 nA in L325H oocytes and 92.2 +/- 12.6 nA in EAAT1 oocytes. These results suggest that even though glutamate and neutral amino acid transporters have strong overall homology, the local structure in the transmembrane domains may be different.     

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.     

Powering the peptide pump: TAP crosstalk with energetic nucleotides

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with āntigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.     

Molecular mechanisms of sugar transport across mammalian and microbial cell membranes

Recent DNA cloning studies have revealed the existence of a large family of homologous sugar transporters in both prokaryotic and eukaryotic organisms. The family includes passive transporters typical of mammalian tissues and active, H(+)-linked sugar transporters from bacteria. Each of these transporters characteristically contains two groups of six putative membrane-spanning alpha-helices separated by a large, hydrophilic, cytoplasmic region. Both the N-terminal and C-terminal regions of the sequence are also predicted to be cytoplasmic. Biophysical and other studies on the human erythrocyte glucose transporter, the only member of the family so far isolated in functional form, suggest that the membrane-spanning alpha-helices associate to form a hydrophilic channel or a substrate-binding cleft extending across the membrane. It is likely that the mechanism of substrate translocation involves alternate exposure of the substrate-binding site to each face of the membrane via a conformational change. Studies in progress on the erythrocyte transporter are beginning to identify regions of the protein involved in substrate binding and the conformational change, and should throw light on the mechanism of sugar translocation in the sugar transporter family as a whole.     

A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines

Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.     

Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus

Catalysis of glutamate transport across cell membranes and coupling of the concentrative transport to sodium, proton, and potassium gradients are processes fundamental to organisms in all kingdoms of life. In bacteria, glutamate transporters participate in nutrient uptake, while in eukaryotic organisms, the transporters clear glutamate from the synaptic cleft. Even though glutamate transporters are crucial to the viability of many life forms, little is known about their structure and quaternary organization. In particular, the subunit stoichiometry of these polytopic integral membrane proteins has not been unequivocally defined. Determination of the native molecular mass of membrane proteins is complicated by their lability in detergent micelles and by their association with detergent and/or lipid molecules. Here we report the purification of glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus in a monodisperse, detergent-solubilized state. Characterization of both transporters either by chemical cross-linking and mass spectrometry or by size-exclusion chromatography and in-line laser light scattering, refractive index, and ultraviolet absorption measurements shows that the transporters have a trimeric quaternary structure. Limited proteolysis further defines regions of primary structure that are exposed to aqueous solution. Together, our results define the subunit stoichiometry of high-affinity glutamate transporters from B. caldotenax and B. stearothermophilus and localize exposed and accessible elements of primary structure. Because of the close amino acid sequence relationship between bacterial and eukaryotic transporters, our results are germane to prokaryotic and eukaryotic glutamate and neutral amino acid transporters.     

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.     

Neuronal glutamate transporters vary in substrate transport rate but not in unitary anion channel conductance

Excitatory amino acid transporters (EAATs) not only sustain a secondary active glutamate transport but also function as anion-selective ion channels. The relative proportion of currents generated by glutamate transport or by the chloride conductance varies for each cloned EAAT subtype. For EAAT1, EAAT2, and EAAT3, the anion current is only a small component of the total transporter-associated current amplitude, whereas EAAT4 and EAAT5 transporters mediate predominantly anion currents. We here demonstrate that the distinct current proportions are entirely due to differences in glutamate transport rates. EAAT3 and EAAT4 differ in unitary glutamate transport rates as well as in the voltage and substrate dependence of anion channel opening, but ion conduction properties are very similar. Noise analysis revealed identical unitary current amplitudes and similar absolute open probabilities for the two anion channels. The low glutamate transport rate of EAAT4 allows regulation of cellular excitability without interfering with extracellular glutamate homeostasis and makes this EAAT isoform ideally suited to regulate excitability in dendritic spines of Purkinje neurons.     

Fast removal of synaptic glutamate by postsynaptic transporters

Glutamate transporters are believed to remove glutamate from the synaptic cleft only slowly because they cycle slowly. However, we show that when glutamate binds to postsynaptic transporters at the cerebellar climbing fiber synapse, it evokes a conformation change and inward current that reflect glutamate removal from the synaptic cleft within a few milliseconds, a time scale much faster than the overall cycle time. Contrary to present models, glutamate removal does not require binding of an extracellular proton, and the time course of transporter anion conductance activation differs from that of glutamate removal. The charge movement associated with glutamate removal is consistent with the majority of synaptically released glutamate being removed from the synaptic cleft by postsynaptic transporters.