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In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na(+) ions and one proton, and the counter-transport of one K(+) ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K(+) ions and the number of Na(+) ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na(+) ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na(+) binding. Mutations to all three proposed Na(+) sites influence the binding of substrate and/or Na(+), or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na(+) affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na(+) and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na(+) and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange.

To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.

We describe an ATP-binding cassette (ABC) transporter in Pseudomonas putida KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for acidic amino acid transport) is encoded by an operon involving genes PP1071-PP1068. A deletion mutant with inactivated solute-binding protein (KTaatJ) failed to grow on Glu and Gln as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KTaatP) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp (K(d)=0.4 and 1.3 muM, respectively), while Gln and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the sigma(54)-dependent two-component system AauRS. Binding of the response regulator AauR to the aat promoter was examined by gel mobility shift assays and DNase I footprinting. By in silico screening, the AauR-binding motif (the inverted repeat TTCGGNNNNCCGAA) was detected in further P. putida KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H(+)/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.

The high-affinity uptake of the acidic amino acid D-aspartate was inhibited in a dose- and time-dependent manner, when C6 cells were exposed to trypsin. The protease decreased the maximal velocity for uptake but not its Km, consistent with a reduction in the number of competent carriers at the plasma membrane. Cellular energy production and [K+]i were unaffected, indicating that the transporter itself was the site of trypsin action. Maximum inhibition of uptake was 50%, which suggests the presence of a heterogeneous population of transporters, only half of which is sensitive to trypsin. These results support our earlier postulate that in glial cells, the high-affinity transporter for acidic amino acids is a transmembrane protein, part of which extends into the external environment.

In vitro cell culture system is one of useful tools for analyzing function of the blood brain barrier (BBB). We have established conditionally immortalized endothelial cell lines from the transgenic rat and mouse harboring temperature sensitive SV40 large T antigen gene. These culture cells expressed GLUT-1 and mdrIa and their growth depended on the expression of is SV40 T antigen in cells. Using these cell lines together with in vivo analyzing system such as brain efflux index method, we revealed following new BBB functions: 1. Amino acid transport involved in osmoregulation. 2. Stereoselective acidic amino acid transport. 3. Efflux transport system of neurotransmitter, GABA. 4. Efflux transport system of neurotransmitter metabolites, homovanillic acid, and effect of uremic toxin on the efflux system. 5. Supply creatine as energy depot to brain. These evidences suggested that the BBB is not only a supporting system but also a protecting system for maintaining the central nervous system.

1. The pharmacological specificity of N-methyl-D-aspartate (NMDA) receptors has been investigated in the rat cerebral cortex by use of radioligand binding and electrophysiological techniques. 2. A comparison was made between a functional assay (NMDA-induced depolarizations in a rat cortical slice preparation) and NMDA-sensitive L-[3H]-glutamate binding in the same brain region and species, to provide accurate affinity values for agonists and antagonists at the NMDA recognition site. 3. In a preparation of crude postsynaptic densities (PSD) from rat cortex, L-[3H]-glutamate bound with high affinity to an NMDA-sensitive population of sites with KD (geometric mean (-s.e.mean. + s.e. mean) = 120 (114, 126) nM, Bmax (mean +/- s.e.mean) = 11.4 +/- 0.8 pmol mg-1 protein and Hill coefficient (mean +/- s.e.mean) = 1.2 +/- 0.17 (n = 3). 4. There was a good agreement between the relative affinities in radioligand binding and electrophysiological assays for the receptor agonists NMDA, N-methyl-L-aspartate, quinolinate and trans-2,3-piperidine dicarboxylate, which are poor substrates of acidic amino acid transport systems. However, agonists which are good substrates for high affinity uptake systems (L- and D-glutamate, L- and D-aspartate, D-aspartate-beta-hydroxamate and L-glutamate-gamma-hydroxamate) were much weaker in the electrophysiological experiments. 5 Schild analysis of the antagonism of NMDA responses in the rat cortical slice by DL-3(2- carboxypiperazin-4-yl)propyl-1-phosphonate, D- and DL-2-amino-5-phosphonovalerate, D- and DL-2- amino- 7-phosphonoheptanoate, D-beta-aspartylaminomethylphosphonate, D-gamma-glutamylglycine and D-Ofaminoadipate (D-AA) indicated a competitive interaction with respective pA2 values of 6.17, 5.62, 5.24, 5.28, 5.20, 5.00, 4.43 and 3.97. 6 In the radioligand binding experiments the same antagonists inhibited only the NMDA-sensitive component of L-[3H]-glutamate binding. IC50 values showed a good correlation with the pA2 values (correlation coefficient = 0.96), with the exception of D-AA which was more potent than anticipated in the binding experiments (IC50 = 9.8 microM).7 These results confirm that NMDA-sensitive L-[3H]-glutamate binding sites represent the NMDA recognition site of the NMDA receptor and provide affinity values for both agonists and antagonists in the rat cerebral cortex, agreeing well with previous estimates in this and other tissues.

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.

The uptake of acidic amino acids by goldfish photoreceptors was investigated by light microscope autoradiography. Isolated retinas were incubated in media containing micromolar amounts of L-[3H]aspartate, L-[3H]glutamate, and D-[3H]aspartate. We have four major observations. (i) Rods accumulate L-[3H]glutamate with a high-affinity transport system; they exhibit a glutamate-to-aspartate selectivity ratio of 30:1. When incubated in 1-10 microM L-[3H]glutamate, rods label more densely than cones. A unit area of rod membrane transports glutamate 30 times better than a unit area of cone membrane. (ii) Red-sensitive and green-sensitive cones show accumulation of L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate, apparently with high affinity, but with little selectivity. Because rods have poor aspartate uptake, red-sensitive and green-sensitive cones may be preferentially labeled with L-[3H]aspartate or D-[3H]aspartate, (iii) Blue-sensitive cones show no uptake of L-[3H]aspartate, D-[3H]aspartate, or L-[3H]glutamate other than that attributable to low-affinity transport. (iv) Various cell types in the goldfish retina can clearly discriminate between glutamate and aspartate, unlike acidic amino acid transport systems described in mammalian brain.

Kinetic studies of uptake demonstrate the existence of an active transport system for d - and l -acidic amino acids in Neurospora crassa. This system, designated amino acid Transport System IV, has K m ' s for l -cysteic, l -aspartic and l -glutamic acids of 7, 13 and 16 μM, respectively. It has higher affinity for d -aspartic acid than for l -aspartic acid but lower affinity for d -glutamic than for l -glutamic acid. Transport System IV has low activity in growing mycelial pads but has higher activity in carbon-, nitrogen- or sulfur-starved pads. The characterization of Transport System IV appears to complete the outlining of the major amino acid transport systems in Neurospora. A comparison is made among the substrate affinities and regulation of several amino transport systems in Neurospora and in two other fungi.

1. 1. l -Aspartate transport activity was optimally induced following 6 h growth on acetate minimal media. 2. 2. The observed aspartate (and glutamate) transport activity was attributed to the general amino acid transport system since l -phenylalanine, but not l -cysteic acid, inhibited transport activity at all pH values tested. 3. 3. Changes in K m values for l -aspartate and l -glutamate transport following growth on acetate minimal media indicated that apparent “affinities” of the “acidic amino acid transport systems” dramatically increased during the stages of active growth.

Summary: Prosthecae purified from cells of Asticcacaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.