The interaction of the gamma-aminobutyric acid transporter GAT-1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive cysteine residue engineered into extracellular loop IV.
The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by cysteine scanning mutagenesis. Fourteen residues of this loop have been replaced by cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible cysteine residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.
Zomot
Elia
E
Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel.
Kanner
Baruch I
BI
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002713:Chlorine / Q000494:pharmacology; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D050485:GABA Plasma Membrane Transport Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008698:Mesylates; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014982:Xenopus laevis / Q000378:metabolism
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D027361:Organic Anion Transporters; D018075:RNA, Complementary; C493413:SLC6A1 protein, human; D013439:Sulfhydryl Reagents; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D002713:Chlorine; D012964:Sodium