Transmembrane domain 8 of the {gamma}-aminobutyric acid transporter GAT-1 lines a cytoplasmic accessibility pathway into its binding pocket.
GAT-1 is a sodium- and chloride-coupled gamma-aminobutyric acid (GABA) transporter, which fulfills an essential role in the synaptic transmission by this neurotransmitter. Cysteine-399 is the major site of inhibition of GAT-1 by membrane-permeant sulfhydryl reagents. This cysteine residue was previously thought to reside on a cytoplasmic loop connecting transmembrane domains (TMs) 8 and 9. However, the crystal structure of LeuT, a bacterial homologue of the mammalian neurotransmitter:sodium symporters, revealed that the residue corresponding to Cys-399 is in fact located in the middle of TM 8. This residue is located to the cytoplasmic side of Asp-395 and Ser-396, whose side chains are thought to ligand one of the two cotransported sodium ions. To determine how the sulfhydryl reagents approach cysteine-399, a cysteine scan of all 35 residues of TM 8 was performed. Sulfhydryl reagents inhibited transport when a cysteine residue was present at either of the positions 399, 402, 406, and 410. SKF-89976A and other non-transportable analogues, which are expected to lock the transporter in a conformation facing the extracellular medium, protected against the sulfhydryl modification at positions 399, 402, and 406. Such a protection was not seen by GABA itself, which actually modestly potentiated the modification at positions 399 and 402. Our results point to an alpha-helical stripe on TM8 lining an aqueous access pathway from the cytoplasm into the binding pocket, which gets occluded in the conformation of the transporter where the binding pocket is exposed to the extracellular medium.
Ben-Yona
Assaf
A
Department of Biochemistry, Hebrew University Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel.
Kanner
Baruch I
BI
D001224:Aspartic Acid / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012694:Serine / Q000737:chemistry; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
D050485:GABA Plasma Membrane Transport Proteins; D001224:Aspartic Acid; D012694:Serine; D005680:gamma-Aminobutyric Acid; D003545:Cysteine