The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue.
The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of neurotransmitter transporters that maintain low synaptic levels of the transmitter. Transport by GAT-1 is sensitive to the polar sulfhydryl reagent 2-aminoethyl methanethiosulfonate. Following replacement of endogenous cysteines to other residues by site-directed mutagenesis, we have identified cysteine 399 as the major determinant of the sensitivity of the transporter to sulfhydryl modification. Cysteine-399 is located in the intracellular loop connecting putative transmembrane domains eight and nine. Binding of both sodium and chloride leads to a reduced sensitivity to sulfhydryl reagents, whereas subsequent binding of GABA increases it. Strikingly binding of the nontransportable GABA analogue SKF100330A gives rise to a marked protection against sulfhydryl modification. These effects were not observed in C399S transporters. Under standard conditions GAT-1 is almost insensitive toward the impermeant 2-(trimethylammonium)ethyl methanethiosulfonate. However, in a chloride-free medium, addition of SKF100330A renders wild type GAT-1, but not C399S, very sensitive to this impermeant reagent. These observations indicate that the accessibility of cysteine 399 is highly dependent on the conformation of GAT-1. Consequently, topological assignments based on accessibility of endogeneous or engineered cysteines to small polar sulfhydryl reagents need to be interpreted with extreme caution.
Golovanevsky
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Department of Biochemistry, Hadassah Medical School, The Hebrew University, P.O. Box 12272, Jerusalem, Israel 91120, USA.
Kanner
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BI
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D009539:Nicotinic Acids / Q000494:pharmacology; D027361:Organic Anion Transporters; D017433:Protein Structure, Secondary; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009539:Nicotinic Acids; D027361:Organic Anion Transporters; C493413:SLC6A1 protein, human; D013439:Sulfhydryl Reagents; C093171:methanethiosulfonate ethylammonium; D005680:gamma-Aminobutyric Acid; C045597:SK&F 100330-A; D005020:Ethyl Methanesulfonate