title: GABA acts as a ligand chaperone in the early secretory pathway to promote cell surface expression of GABAA receptors.
abstract: GABA (gamma-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABA(A) receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABA(A) receptor cell surface expression. Forty-two hours of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GABA-gated chloride currents. In time-course experiments, a 1h GABA exposure, followed by a 5h incubation in GABA-free medium, was sufficient to increase receptor surface expression. A shorter GABA exposure could be used in HEK 293 cells stably transfected with the GABA transporter GAT-1. In rGAT-1HEK 293 cells, the GABA effect was blocked by the GAT-1 inhibitor NO-711, indicating that GABA was acting intracellularly. The effect of GABA was prevented by brefeldin A (BFA), an inhibitor of early secretory pathway trafficking. Coexpression of GABA(A) receptors with the GABA synthetic enzyme glutamic acid decarboxylase 67 (GAD67) also resulted in an increase in receptor surface levels. GABA treatment failed to promote the surface expression of GABA binding site mutant receptors, which themselves were poorly expressed at the surface. Consistent with an intracellular action of GABA, we show that GABA does not act by stabilizing surface receptors. Furthermore, GABA treatment rescued the surface expression of a receptor construct that was retained within the secretory pathway. Lastly, the lipophilic competitive antagonist (+)bicuculline promoted receptor surface expression, including the rescue of a secretory pathway-retained receptor. Our results indicate that a neurotransmitter can act as a ligand chaperone in the early secretory pathway to regulate the surface expression of its receptor. This effect appears to rely on binding site occupancy, rather than agonist-induced structural changes, since chaperoning is observed with both an agonist and a competitive antagonist.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center-Shreveport, 1501 Kings Highway, Shreveport, LA 71130-3932, USA.
authors.forename: Randa S
authors.initials: RS
authors.lastname: Eshaq
authors.forename: Letha D
authors.initials: LD
authors.lastname: Stahl
authors.forename: Randolph
authors.initials: R
authors.lastname: Stone
authors.forename: Sheryl S
authors.initials: SS
authors.lastname: Smith
authors.forename: Lucy C
authors.initials: LC
authors.lastname: Robinson
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Leidenheimer
mesh_terms: D001640:Bicuculline / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000502:physiology; D005434:Flow Cytometry; D018756:GABA Antagonists / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism; D058787:GABA-A Receptor Antagonists; D005968:Glutamate Decarboxylase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008024:Ligands; D018832:Molecular Chaperones / Q000502:physiology*; D009154:Mutation / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000187:drug effects; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000187:drug effects; D005680:gamma-Aminobutyric Acid / Q000502:physiology*
chemical_list: D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D058787:GABA-A Receptor Antagonists; D008024:Ligands; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D020126:Brefeldin A; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D001640:Bicuculline