Proximity of transmembrane domains 1 and 3 of the γ-aminobutyric acid transporter GAT-1 inferred from paired cysteine mutagenesis

GAT-1 is a sodium- and chloride-dependent γ-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, ∼70% inhibition of transport was observed by cadmium with an IC₅₀ of ∼10 μM. This inhibition was not observed in the corresponding single mutants and also not in >10 other double mutants, except for V67C/I143C, where the half-maximal effect was obtained at ∼50 μM. 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.