Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1.
The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.
Zhou
Yonggang
Y
Department of Biochemistry, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Zomot
Elia
E
Department of Biochemistry, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Kanner
Baruch I
BI
Department of Biochemistry, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel. Electronic address: kannerb@cc.huji.ac.il.
D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008094:Lithium / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D050485:GABA Plasma Membrane Transport Proteins; C493413:SLC6A1 protein, human; D005680:gamma-Aminobutyric Acid; D008094:Lithium; D012964:Sodium