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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.

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.

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.

By selection on a medium containing both ethionine and p-fluorophenylalanine a mutant strain of Neurospora has been isolated. This strain, nap, shows greatly decreased transport of neutral and acidic amino acids. The mutation also confers resistance to 4-methyltryptophan, aminoprotein and glycine. The physiological defect appears to be specific since the mutant showed a normal doubling time for protein, normal oxygen consumption and normal transport of glucose, sulfate and basic amino acids. This gene represents a third locus which governs neutral and acidic amino acid transport in Neurospora, the two previously described being un-t (55701) and mtr.

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.

The uptake of l -glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of l -glutamate uptake has provided information as to the substrate specificity and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting l -glutamate and aspartate and certain closely related acidic amino acid analogues.

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.

Penicillium chrysogenum possesses a relatively specific constitutive transport system for the basic amino acids l -lysine and l -arginine (Km = 6 × 10−6 m , Vmax = 1 μmole/g-min). This system has a low affinity for 2,4-diaminobutryate, l -ornithine, and l -histidine. The basic amino acid transport system can be assayed readily in nutrient-sufficient mycelia where a nonspecific amino acid transport system (Benko, P. V., Wood, T. C., and Segel, I. H., Arch. Biochem. Biophys.129, 498, 1969), which also transports basic amino acids, is extremely low in activity. Nutrient-sufficient mycelia also contain a low-level lysine-insensitive arginine transport system and a low-level arginine-insensitive lysine transport system. The basic amino acid transport system can be assayed in nitrogen-starved and in carbon-starved mycelia if a large excess of, e.g., l -leucine is added to the assay mixture in order to suppress transport of the labeled substrate by the nonspecific amino acid transport system. The acidic amino acids, l -glutamate and l -aspartate, are also substrates of the general amino acid transport system. However, the preferred ionic form is the uncharged species. l -Glutamate and l -aspartate are also transported by a distinct stereospecific acidic amino acid transport system that develops along with the nonspecific system during nitrogen starvation or carbon starvation. The acidic amino acid system can be assayed at pH 6.0 in the presence of a large excess of, e.g., l -leucine. These conditions minimize the transport of glutamate and aspartate by the nonspecific system. A distinct proline transport system has also been detected in nitrogen-starved and in carbon-starved mycelia. No distinct or quantitatively significant specific systems for aromatic amino acids or histidine were detected. The general and acidic amino acid transport systems are subject to feedback inhibition (“transinhibition”) by their intracellular substrates. The transinhibition is most evident under conditions in which the amino acid is transported much faster than it is metabolized. In this respect, l -α-aminoadipate is a better transinhibitor of the acidic system than the preferred substrates, aspartate and glutamate. The feedback inhibitor of the nonspecific system has been shown to be the accumulated substrate rather than NH4+.

nap , previously characterized by Jacobson and Metzenberg as a neutral and acidic amino acid transport mutant, was found in this study to be reduced in transport activity for a variety of metabolites. Analysis of glycoprotein molecules involved in amino acid binding indicates that the deficiency is not at this level. The deficiency appears to be in some common component of all active transport systems.