Proximity of Transmembrane Domains 1 and 3 of the γ-AminobutyricAcid Transporter GAT-1 Inferred from Paired CysteineMutagenesis

Abstract

GAT-1 is a sodium-and chloride-dependent ␥-aminobutyric acid transporter and is the first identified member of a family of transporters that maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. Because transmembrane domains 1 and 3 contain amino acid residues important for transport activity, we hypothesized that these domains may participate in the formation of the binding pocket of the transporter. Pairwise substitutions have been introduced in several predicted transmembrane domains and in the first extracellular loop of GAT-1. In the double mutant W68C/I143C, in which the cysteines were introduced at locations at the extracellular part of transmembrane domains 1 and 3, respectively, ϳ70% inhibition of transport was observed by cadmium with an IC 50 of ϳ10 M. This inhibition was not observed in the corresponding single mutants and also not in >10 other double mutants, except for V67C/I143C, where the halfmaximal effect was obtained at ϳ50 M. The inhibition by cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Our results suggest that transmembrane domains 1 and 3 come in close proximity within the transporter monomer.The overall process of synaptic transmission is terminated by neurotransmitter transporters located in the plasma membranes of cells surrounding the synapse. Most neurotransmitters are removed from the synaptic cleft by sodium-and chloridedependent transporters, which form a family that also includes (besides the transporters for ␥-aminobutyric acid (GABA)) 1 those for serotonin, dopamine, norepinephrine, and glycine (for reviews, see Refs. 1 and 2). The GABA transporter GAT-1 (3, 4), the first identified member of this family, catalyzes electrogenic sodium:chloride:GABA cotransport with a stoichiometry of 2:1:1 (5-8). The role of chloride in this process is still under debate, because it has been proposed that, during sodiumcoupled GABA transport, obligatory chloride out /chloride in exchange takes place (9). The predicted topology of GAT-1 (4) and the other members of this family is 12 TMDs linked by hydrophilic loops with the amino and carboxyl termini located inside the cell, and strong experimental support for this model has been obtained using the serotonin transporter SERT (10).GAT-1 has 15 endogenous cysteine residues of which three are located on extracellular loops. Studies on the related dopamine and serotonin transporters indicate that the cysteine residues, equivalent to their GAT counterparts at positions 164 and 173 (which are located in the second extracellular loop), form a disulfide bond (11,12). This leaves cysteine 74, located on the first extracellular loop, as the only cysteine that reacts with impermeant methanethiosulfonate reagents. GABA transport by wild-type GAT-1 is only very modestly inhibited by the membrane-impermeant (2-(trimethylammonium) ethyl-)methanethiosulfonate (13), indicating that this position is not easily accessible. The reagent (2-aminoethyl)methanethiosulfonate has a d...

Tyr-95 and Ile-172 in Transmembrane Segments 1 and 3 of Human Serotonin Transporters Interact to Establish High Affinity Recognition of Antidepressants

“…However, the position of the hSERT D98 orthologous residue in LeuTAa strongly supports our idea that Asp-98 contributes to the formation of the cadmium-binding site. These data are in agreement with a recent report using this methodology to provide evidence of proximity of TM1 and TM3 in the tertiary structure of a transporter with the same family as SERT, GAT1 (25).…”

The binding sites for cocaine and dopamine in the dopamine transporter overlap

“…In addition, these positions have been cross-linked in the related GAT-1 (ref. 34). We observed a >threefold decrease in [ 3 H]CFT off-rate on treatment of the resulting double mutant E2C W84C-I159C with 0.5 mM MTS-3-MTS after prebinding of [ 3 H]CFT (Fig.…”

Substrates and Non-transportable Analogues Induce Structural Rearrangements at the Extracellular Entrance of the Glial Glutamate Transporter GLT-1/EAAT2

“…Inhibition by Copper(II) (1, 3 -This was basically done as described (24,25). HeLa cells transfected with the indicated construct were washed once with choline solution (150 mM choline chloride, 5 mM KP i , pH 7.4, 0.5 mM MgSO 4 , and 0.3 mM CaCl 2 ) and preincubated with the solutions of different compositions containing the indicated concentrations of CuPh.…”