The Membrane Topology of GAT-1, a (Na++ Cl−)-coupled γ-Aminobutyric Acid Transporter from Rat Brain

The membrane topology of GAT-1, a sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain, has been probed using N-glycosylation scanning mutagenesis. Overall, the results support the theoretical 12-transmembrane segment model. This model (based on hydropathy analysis) was originally proposed for GAT-1 and adopted for all other members of the sodium- and chloride-dependent neurotransmitter transporter superfamily. However, our data indicate that the loop connecting putative transmembrane domains 2 and 3, which was predicted to be located intracellularly, can be glycosylated in vivo. Furthermore, studies with permeant and impermeant methanesulfonate reagents suggest that cysteine 74, located in the hydrophilic loop connecting transmembrane domains 1 and 2, is intracellular rather than extracellular. We present a model in which the topology deviates from the theoretical one in the amino-terminal third of the transporter. It also contains 12 transmembrane segments, but the highly conserved domain 1 does not form a conventional transmembrane alpha-helix.