The substrates of the gamma-aminobutyric acid transporter GAT-1 induce structural rearrangements around the interface of transmembrane domains 1 and 6.
The sodium- and chloride-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 is essential for efficient synaptic transmission by this neurotransmitter. GAT-1 is the first cloned member of the neurotransmitter-sodium-symporter family. Here we address the idea that during transport the extracellular halves of transmembrane domains (TM) 1 and 6, TM 1b/TM 6a, move relative to the binding pocket. Therefore, we have probed the aqueous accessibility of TM 6a and its proximity to TM 1b in the presence and absence of its substrates. Cysteines were introduced, one by one, at all TM 6a positions. In several mutants, transport activity was inhibited by the impermeant sulfhydryl reagent (2-trimethylammonium)methanethiosulfonate, whereas wild type GAT-1 was basically insensitive. This inhibition was potentiated by sodium, whereas GABA was protective. Moreover, we used paired cysteine mutagenesis in conjunction with treatments with copper(II)(1,10-phenanthroline)(3) (CuPh). CuPh did not affect the activity of wild type GAT-1 but potently inhibited transport by the TM 6a mutant D287C. Such inhibition was not observed with D287C/C74A, indicating that Asp-287 is close to Cys-74 of TM 1b. Inhibition of transport of D287C by CuPh, but not by (2-trimethylammonium)methanethiosulfonate, was potentiated when sodium and GABA were both removed. Thus, the degree of inhibition by CuPh is not a simple function of the accessibility of the individual cysteines but also involves structural rearrangements around the TM 1b/TM 6a interface.
Rosenberg
Alex
A
Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem, Israel.
Kanner
Baruch I
BI
D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007501:Iron / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D050485:GABA Plasma Membrane Transport Proteins; D013438:Sulfhydryl Compounds; D002104:Cadmium; D007501:Iron; D003545:Cysteine