Transporter-associated currents in the gamma-aminobutyric acid transporter GAT-1 are conditionally impaired by mutations of a conserved glycine residue.
To determine whether glycine residues play a role in the conformational changes during neurotransmitter transport, we have analyzed site-directed mutants of the gamma-aminobutyric acid (GABA) transporter GAT-1 in a domain containing three consecutive glycines conserved throughout the sodium- and chloride-dependent neurotransmitter transporter family. Only cysteine replacement of glycine 80 resulted in the complete loss of [(3)H]GABA uptake, but oocytes expressing this mutant exhibited the sodium-dependent transient currents thought to reflect a charge-moving conformational change. When sodium was removed and subsequently added back, the transients by G80C did not recover, as opposed to wild type, where recovery was almost complete. Remarkably, the transients by G80C could be restored after exposure of the oocytes to either GABA or a depolarizing pre-pulse. These treatments also resulted in a full recovery of the transients by the wild type. Whereas in wild type lithium leak currents are observed after prior sodium depletion, this was not the case for the glycine 80 mutants unless GABA was added or the oocytes were subjected to a depolarizing pre-pulse. Thus, glycine 80 appears essential for conformational transitions in GAT-1. When this residue is mutated, removal of sodium results in "freezing" the transporter in one conformation from which it can only exit by compensatory changes induced by GABA or depolarization. Our results can be explained by a model invoking two outward-facing states of the empty transporter and a defective transition between these states in the glycine 80 mutants.
Zhou
Yonggang
Y
Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel.
Kanner
Baruch I
BI
D000818:Animals; D019921:Biotinylation; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D004553:Electric Conductivity; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D005998:Glycine / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008094:Lithium / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection; D014316:Tritium; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; D008698:Mesylates; D018075:RNA, Complementary; C493413:SLC6A1 protein, human; D013439:Sulfhydryl Reagents; D014316:Tritium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005680:gamma-Aminobutyric Acid; D008094:Lithium; D012964:Sodium; D003545:Cysteine; D005998:Glycine