Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na ؉ followed by countertransport of K ؉ . Two Tl ؉ binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K ؉ -loaded liposomes was impaired. However, with Na ؉ and unlabeled L-aspartate inside the liposomes, exchange levels were around 50 -90% of those by wild-type. In further contrast to wild-type, where either substrate or K ؉ stimulated the anion conductance by the transporter, substrate but not K ؉ modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.cation binding site ͉ obligate exchange mutant ͉ sodium selectivity G lutamate transporters are key elements in the termination of the synaptic actions of the neurotransmitter and keep its synaptic concentrations below neurotoxic levels. Glutamate transport is an electrogenic process (1, 2) consisting of two sequential translocation steps: (i) Cotransport of the neurotransmitter with three sodium ions and a proton (3, 4) and (ii) the countertransport of one potassium ion (5-7). The mechanism involving two half-cycles (Fig. 1A) is supported by the fact that mutants impaired in the interaction with potassium are ''locked'' in an obligatory exchange mode (7,8). Glutamate transporters mediate two distinct types of substrate-induced steady-state current: An inward-rectifying or ''coupled'' current, reflecting electrogenic sodium-coupled glutamate translocation, and an ''uncoupled'' sodium-dependent current, which is carried by chloride ions and further activated by substrates of the transporter (9-11). Nontransportable substrate analogs, expected to ''lock'' the transporter in an outward-facing conformation (stippled part of Fig. 1 A), are not only competitive inhibitors of the two types of substrate-induced current, but also inhibit the basal sodium-dependent anion conductance (12,13).Recently a high-resolution crystal structure of a glutamate transporter homolog, Glt Ph , from the archeon Pyrococcus horikoshii was pu...
In the central nervous system, electrogenic sodium- and potassium-coupled glutamate transporters terminate the synaptic actions of this neurotransmitter. In contrast to acidic amino acids, dicarboxylic acids are not recognized by glutamate transporters, but the related bacterial DctA transporters are capable of transporting succinate and other dicarboxylic acids. Transmembrane domain 8 contains several residues that differ between these two types of transporters. One of these, aspartate-444 of the neuronal glutamate transporter EAAC1, is conserved in glutamate transporters, but a serine residue occupies this position in DctA transporters. When aspartate-444 is mutated to serine, cysteine, alanine, or even to glutamate, uptake of d-[3H]-aspartate as well as the inwardly rectifying steady-state currents induced by acidic amino acids is impaired. Even though succinate was not capable of inducing any steady-state transport currents, the dicarboxylic acid inhibited the sodium-dependent transient currents by the mutants with a neutral substitution at position 444. In the neutral substitution mutants inhibition of the transients was also observed with acidic amino acids. In the D444E mutant, acidic amino acids were potent inhibitors of the transient currents, whereas the apparent affinity for succinate was lower by at least three orders of magnitude. Even though L-aspartate could bind to D444E with a high apparent affinity, this binding resulted in inhibition rather than stimulation of the uncoupled anion conductance. Thus, a carboxylic acid–containing side chain at position 444 prevents the interaction of glutamate transporters with succinate, and the presence of aspartate itself at this position is crucial for productive substrate binding compatible with substrate translocation.
Background:The cation binding sites of brain glutamate transporters are not yet identified. Results: Mutation of a conserved asparagine residue changed cation selectivity and apparent substrate affinity. Conclusion: This residue plays a crucial role in the ion-coupling mechanism of glutamate transporters. Significance: The proposed direct coupling of the cation and solute fluxes may be relevant for other ion-coupled transporters.
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