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Neurotransmitter:sodium symporters are crucial for efficient synaptic transmission. The transporter GAT-1 mediates electrogenic cotransport of GABA, sodium, and chloride. The presence of chloride enables the transporter to couple the transport of the neurotransmitter to multiple sodium ions, thereby enabling its accumulation against steep concentration gradients. Here we study the functional impact of mutations of the putative chloride-binding residues on transport by GAT-1, with the emphasis on a conserved glutamine residue. In contrast to another putative chloride coordinating residue, Ser-331, where mutation to glutamate led to chloride-independent GABA transport, the Q291E mutant was devoid of any transport activity, despite substantial expression at the plasma membrane. Low but significant transport activity was observed with substitution mutants with small side chains such as Q291S/A/G. Remarkably, when these mutations were combined with the S331E mutation, transport was increased significantly, even though the activity of the S331E single mutant was only ∼25% of that of wild type GAT-1. Transport by these double mutants was largely chloride-independent. Like mutants of other putative chloride coordinating residues, the apparent affinity of the active Gln-291 single mutants for chloride was markedly reduced along with a change their anion selectivity. In addition to the interaction of the transporter with chloride, Gln-291 is also required at an additional step during transport. Electrophysiological analysis of the Q291N and Q291S mutants, expressed in Xenopus laevis oocytes, is consistent with the idea that this additional step is associated with the gating of the transporter.

Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a beta-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step.

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b(o,+) exchanges dibasic for neutral amino acids. It is composed of rBAT and b(o,+)AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. b(o,+)AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the b(o,+) substrates inside the liposomes. rBAT was essential for the cell surface expression of b(o,+)AT, but it was not required for reconstituted b(o,+)AT transport activity. No system b(o,+) transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted b(o,+)AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of b(o,+)AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted b(o,+)AT activity. Thus, subunit b(o,+)AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.

Two high affinity Zn2+ binding sites were engineered in the otherwise Zn2+-insensitive rat γ-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn2+ 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 Zn2+ (IC50 = 35 and 44 μm, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn2+ was a potent inhibitor of the GABA-induced current (IC50 = 21 μm for T349H/E370H and 51 μm for T349H/Q374C), albeit maximum inhibition was only ∼40% in T349H/E370H versus ∼90% in T349H/Q374C. In the wild type, Zn2+ 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 Zn2+ caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn2+ 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 Zn2+ 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.

The sodium and chloride coupled γ-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 α-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.

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.

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.

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.

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.

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.

Nature 360, 464-467 (1992) THE third author's name was shown incorrectly in this letter. It should read Magnar Bj0ras, as above. In addition, Fig. 2b is not, as stated, a western blot but is an autoradiogram of an SDS-polyacrylamide gel electrophoresis gel.

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.

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.