The membrane topology of GAT-1, a (Na+ + Cl-)-coupled gamma-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.
Bennett
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ER
Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
Kanner
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BI
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000378:metabolism*; D016384:Consensus Sequence; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters; D051381:Rats; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D007202:Indicators and Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; C493415:Slc6a1 protein, rat; C093171:methanethiosulfonate ethylammonium; D005680:gamma-Aminobutyric Acid; D005020:Ethyl Methanesulfonate