Molecular characterization of substrate-binding sites in the glutamate transporter family.

Glutamate transporters are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentrations below neurotoxic levels. These transporters also mediate a thermodynamically uncoupled chloride flux that is activated by two of the molecules that they transport - sodium and glutamate. Five eukaryotic glutamate transporters have been cloned and identified. They exhibit approximately 50% identity and this homology is even greater in the carboxyl terminal half, which is predicted to have an unusual topology. Determination of the topology shows that the carboxyl terminal part of the molecule contains several transmembrane domains that are separated by at least two re-entrant loops. In these structural elements, we have identified several conserved amino acid residues that play crucial roles in the interaction with the transporter substrates sodium, potassium and glutamate.

Engineered Zn(2+) switches in the gamma-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents.

Two high affinity Zn(2+) binding sites were engineered in the otherwise Zn(2+)-insensitive rat gamma-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn(2+) binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [3H]GABA uptake by Zn(2+) (IC(50) = 35 and 44 microM, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn(2+) was a potent inhibitor of the GABA-induced current (IC(50) = 21 microM for T349H/E370H and 51 microM for T349H/Q374C), albeit maximum inhibition was only approximately 40% in T349H/E370H versus approximately 90% in T349H/Q374C. In the wild type, Zn(2+) did not affect the Na(+)-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na(+) binding. However, in both mutants Zn(2+) caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn(2+) is inhibiting transporter function by stabilizing the outward-facing Na(+)-bound state. Translocation of lithium by the transporter does not require GABA binding and analysis of this uncoupled Li(+) conductance revealed a potent inhibition by Zn(2+) in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes of the transporter.

Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter.

Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is cotransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact with potassium are only capable of catalyzing electroneutral exchange. Here we identify a residue involved in controlling substrate recognition in the neuronal transporter EAAC-1 that transports acidic amino acids as well as cysteine. When arginine 447, a residue conserved in all glutamate transporters, is replaced by cysteine, transport of glutamate or aspartate is abolished, but sodium-dependent cysteine transport is left intact. Analysis of other substitution mutants shows that the replacement of arginine rather than the introduced cysteine is responsible for the observed phenotype. In further contrast to wild type, acidic amino acids are unable to inhibit cysteine transport in R447C-EAAC-1, indicating that the selectivity change is manifested at the binding step. Electrophysiological analysis shows that in the mutant cysteine, transport has become electroneutral, and its interaction with the countertransported potassium is impaired. Thus arginine 447 plays a pivotal role in the sequential interaction of acidic amino acids and potassium with the transporter and, thereby, constitutes one of the molecular determinants of coupling their fluxes.

Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology.

In the central nervous system, (Na+ + K+)-coupled glutamate transporters restrict the neurotoxicity of this transmitter and limit the duration of synaptic excitation at some synapses. The various isotransporters exhibit a particularly high homology in an extended hydrophobic domain of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination of the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteines was engineered. Their topological disposition was determined by using a biotinylated sulfhydryl reagent. The glutamate transporter has eight transmembrane domains long enough to span the membrane as et heiices. Strikingly, between the seventh and eighth domains, a structure reminiscent of a pore loop and an outward-facing hydrophobic linker are positioned.

Molecular determinant of ion selectivity of a (Na+ + K+)-coupled rat brain glutamate transporter.

Glutamate transporters remove this neurotransmitter from the synaptic cleft by a two-stage electrogenic process, in which glutamate is first cotransported with three sodium ions and a proton. Subsequently, the cycle is completed by translocation of a potassium ion in the opposite direction. Recently, we have identified an amino acid residue of the glutamate transporter GLT-1 (Glu-404) that influences potassium coupling. We have now analyzed the effect of seven other amino acid residues in the highly conserved region surrounding this site. One of these residues, Tyr-403, also proved important for potassium coupling, because mutation to Phe (Y403F) resulted in an electroneutral obligate exchange mode of glutamate transport. This mutation in the transporter also caused an approximately 8-fold increase in the apparent sodium affinity, with no change in the apparent affinity for L-glutamate or D-aspartate. Strikingly, although exchange catalyzed by the wild-type transporter is strictly dependent on sodium, the selectivity of Y403F mutant transporters is altered so that sodium can be replaced by other alkaline metal cations including lithium and cesium. These results indicate the presence of interacting sites in or near the transporter pore that control selectivity for sodium and potassium.

Mutation of an amino acid residue influencing potassium coupling in the glutamate transporter GLT-1 induces obligate exchange.

Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. After cotransport of glutamic acid with Na+, the cycle is completed by countertransport of K+. We have identified an amino acid residue (glutamate 404) influencing ion coupling in a domain of the transporter implicated previously in kainate binding. Mutation of this residue to aspartate (E404D) prevents both forward and reverse transport induced by K+. Sodium-dependent transmitter exchange and a transporter-mediated chloride conductance are unaffected by the mutation, indicating that this residue selectively influences potassium flux coupling. The results support a kinetic model in which sodium and potassium are translocated in distinct steps and suggest that this highly conserved region of the transporter is intimately associated with the ion permeation pathway.

Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1.

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.

The number of amino acid residues in hydrophilic loops connecting transmembrane domains of the GABA transporter GAT-1 is critical for its function.

Transporter proteins consist of multiple transmembrane domains connected by hydrophillic loops. As the importance of these loops in transport processes is poorly understood, we have studied this question using the cDNA coding for GAT-1, a Na+/Cl(-)-coupled gamma-aminobutyric acid transporter from rat brain. Deletions of randomly picked non-conserved single amino acids in the loops connecting helices 7 and 8 or 8 and 9 result in inactive transport upon expression in HeLa cells. However, transporters where these amino acids are replaced with glycine retain significant activity. The expression level of the inactive mutant transporters was similar to that of the wild-type, but one of these, delta Val-348, appears to be defectively targetted to the plasma membrane. Our data are compatible with the idea that a minimal length of the loops is required, presumably to enable the transmembrane domains to interact optimally with each other.

Phosphorylation and modulation of brain glutamate transporters by protein kinase C.

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.

Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity.

The gamma-aminobutyric acid (GABA) transporter (subtype A) is located in nerve terminals and catalyses coupled electrogenic uptake of the neurotransmitter with two or three sodium and one chloride ions. It contains 599 amino acids and 12 putative membrane spanning alpha-helices and is the first described member of a neurotransmitter transporter superfamily. The membrane domain contains 5 charged amino acids which are basically conserved. Using site-directed mutagenesis, we show that only one of them, arginine 69, is absolutely essential for activity. It is located in a highly conserved region encompassing parts of helices 1 and 2. The three other positively charged amino acids and the only negative charged one, glutamate 467, are not critical. These results suggest that the translocation pathway of the sodium ions through the membrane does not involve charged amino acid residues and underline the importance of the highly conserved stretch between amino acids 66 and 86.

Identification of domains of a cloned rat brain GABA transporter which are not required for its functional expression.

The sodium and chloride coupled gamma-aminobutyric acid (GABA) transporter purified from rat brain, belongs to a superfamily of neurotransmitter transporters. They are involved in the termination of synaptic transmission and are predicted to have 12 membrane spanning alpha-helices with both amino- and carboxyl-termini oriented toward the cytoplasm. In order to define the domains not required for functional expression, we have constructed and expressed a series of deletion mutants in GAT-1, the cDNA clone encoding for the transporter. Transporters truncated at either end until just a few amino-acids distance from the beginning of helix 1 and the end of helix 12, retain their ability to catalyze sodium and chloride-dependent GABA transport.

Cloning and expression of a rat brain L-glutamate transporter.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Two pharmacologically distinct sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporters are present in plasma membrane vesicles and reconstituted preparations from rat brain.

Electrogenic sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transport in crude synaptosomal membrane vesicles is partly inhibited by saturating levels of either of the substrate analogues cis-3-aminocyclohexanecarboxylic acid (ACHC) or beta-alanine. However, both of them together potently and fully inhibit the process. Transport of beta-alanine, which exhibits an apparent Km of about 44 microM, is also electrogenic and sodium and chloride dependent and competitively inhibited by GABA with a Ki of about 3 microM. This value is very similar to the Km of 2-4 microM found for GABA transport. On the other hand, ACHC does not inhibit beta-alanine transport at all. Upon solubilization of the membrane proteins with cholate and fractionation with ammonium sulfate, a fraction is obtained which upon reconstitution into proteoliposomes exhibits 4- to 10-fold-increased GABA transport. This activity is fully inhibited by low concentrations of ACHC and is not sensitive at all to beta-alanine. GABA transport in this preparation exhibits an apparent Km of about 2.5 microM and it is competitively inhibited by ACHC (Ki approximately 7 microM). These data indicate the presence of two GABA transporter subtypes in the membrane vesicles: the A type, sensitive to ACHC, and the B type, sensitive to beta-alanine.

Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.

Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.

Transport of 5-hydroxytryptamine in membrane vesicles from rat basophilic leukemia cells.

Rat basophilic leukemia (RBL) cells were grown as tumors. Membrane vesicles were isolated from them and serotonin transport was measured. Two types of transport were identified. One was sensitive to imipramine and sodium-dependent, while the other was sensitive to reserpine and ATP-dependent. The transport systems exhibit different affinities for serotonin (sodium-dependent, Km 0.22 microM; ATP-dependent, Km 2.6 microM) and are different in their substrate specificity, the former being much more specific. The 5-hydroxytryptamine transport by the reserpine-sensitive system was strongly inhibited by other biogenic amines, like norepinephrine, epinephrine and dopamine, whereas that by the imipramine-sensitive system was not. Upon Ficoll gradient centrifugation the two transport systems were separated. The reserpine-sensitive activity was found much further into the gradient than the imipramine-sensitive one. The latter co-migrated with the receptor of IgE, which is located in the plasma membrane. Characterization of latter showed that in addition to the dependence of 5-hydroxytryptamine influx on external sodium it was also absolutely dependent on external chloride and was strongly stimulated by internal potassium. On the other hand, efflux required external potassium. An alternative potassium independent way of loss of labelled 5-hydroxytryptamine was by exchange. A small but consistent stimulation of influx was observed in the presence of valinomycin, indicating that the process is electrogenic. The reserpine-sensitive system could also be driven in the absence of ATP. This required the imposition of pH gradient (acid inside) and was stimulated by an artificially imposed membrane potential (positive inside).

Efflux and exchange of gamma-aminobutyric acid and nipecotic acid catalysed by synaptic plasma membrane vesicles isolated from immature rat brain.

The mechanism of gamma-aminobutyric acid translocation in synaptic plasma membrane vesicles from rat brain has been probed by comparing the ion dependency of net efflux with that of exchange. Furthermore the question has been asked if the same mechanism operates for other solutes translocated by this transporter. Dilution-induced efflux of gamma-aminobutyrate from the membrane vesicles is about 3-fold stimulated by externally added gamma-aminobutyrate. Half maximal stimulation is obtained at a gamma-aminobutyrate concentration similar to the Km for gamma-aminobutyrate influx. This stimulation (exchange) is dependent on external sodium but not on external chloride. In contrast to this gamma-aminobutyrate influx is absolutely dependent on the simultaneous presence of sodium and chloride ions (Kanner, B.I. (1978) Biochemistry 17, 1207-1211), while efflux is dependent on the presence of these two ions on the inside (Kanner, B.I. and Kifer, L. (1981) Biochemistry 20, 3354-3358). Nigericin stimulates dilution-induced efflux of gamma-aminobutyrate from potassium loaded vesicles to a larger extent than external gamma-aminobutyrate. gamma-Aminobutyrate further enhances the nigericin-induced stimulation, provided that the vesicles are not preloaded with chloride. Nipecotic acid is transported with the same features as gamma-aminobutyrate and the two solutes behave similar with respect to the ion dependence of net flux and exchange. A model for the translocation cycle is proposed in which at least one of the translocated sodium ions binds to the transporter in its 'outside' conformation after chloride and the solute have bound previously. Conversely, the solute is released from its 'inside' conformation prior to chloride and at least one of the sodium ions.

Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain.

Efflux of L-glutamic acid from synaptic plasma membrane vesicles requires external potassium. This requirement is saturated by concentrations of about 15 mequiv/L potassium. In the absence of potassium, L-glutamic acid can be released from the vesicles in the presence of external L-glutamic acid. This stimulation does not require external sodium but is dependent on the external concentration of L-glutamic acid. Half-maximal effects are obtained by concentrations of about 1 microM which are very similar to the apparent Km for L-glutamic acid influx. Efflux of labeled glutamate driven by external sodium plus glutamate requires internal sodium. These findings suggest that the transporter displays an asymmetric behavior toward sodium. This ion dissociates much more slowly than L-glutamic acid on the external surface of the membrane but not on the internal surface. Furthermore, it appears that the transporter translocates potassium in a step distinct from the L-glutamic acid translocation step. The simplest explanation is that upon translocation of sodium and L-glutamic acid and their release to the inside, potassium binds to the transporter, enabling it to return to the outside to allow initiation of a new transport cycle.