Syntaxin 1A inhibits GABA flux, efflux, and exchange mediated by the rat brain GABA transporter GAT1.
GABA transporters control extracellular GABA levels by coupling transmitter uptake to the sodium and chloride cotransport. The rat brain GABA transporter GAT1 and other members of this family are regulated by direct interactions with syntaxin 1A, a protein involved in vesicle docking and in the regulation of several ion channels and transporters. We have shown previously that syntaxin 1A exerts its effects on GAT1 by decreasing the net uptake of GABA and its associated ions through interactions with aspartic acid residues in the N-terminal tail of GAT1. This reduction in net uptake could be caused by many steps in the transport cycle, including substrate binding, substrate flux, substrate efflux, or reorientation of the unliganded transporter. To address this question, we performed GABA flux assays, measured flux- and efflux-associated ion currents, and assessed GABA exchange in multiple experimental systems expressing syntaxin 1A and wild-type GAT1 or GAT1 mutants. Syntaxin 1A caused similar reductions in forward and reverse transport that did not involve changes in apparent transport affinities for sodium, chloride, or GABA. The syntaxin 1A-mediated reduction in GABA flux and efflux was mimicked by mutations in GAT1 at the syntaxin 1A binding site. The binding site GAT1 mutant also caused a reduction in exchange. These data suggest that syntaxin 1A exerts its effects, directly or indirectly, on GAT1 function through interactions with GAT1's N-terminal tail and that the inhibition occurs at a step in the translocation process after substrate binding but which involves both unidirectional transport and transmitter exchange.
D000818:Animals; D000954:Antigens, Surface / Q000494:pharmacology*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D027361:Organic Anion Transporters; D051381:Rats; D050827:Syntaxin 1; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000954:Antigens, Surface; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; C493415:Slc6a1 protein, rat; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D005680:gamma-Aminobutyric Acid