Rat beta 3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of gamma-aminobutyric acid type A receptors.
Human embryonic kidney 293 cells transiently transfected with beta 3 subunits of gamma-aminobutyric acid type A receptors from the rat exhibited a specific high affinity binding for [35S]t-butylbicyclophosphorothionate (TBPS) that could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864. The potency of these compounds for inhibition of [35S]TBPS binding was similar in membranes from beta 3 subunit-transfected human embryonic kidney 293 cells and in cerebellar membranes. In contrast to maximally inhibiting concentrations of unlabeled TBPS or picrotoxin, which caused a monophasic and rather slow dissociation of [35S]TBPS, maximally inhibiting concentrations of pentobarbital, etazolate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864 accelerated the dissociation of [35S]TBPS from beta 3 subunit-containing membranes. Immunoaffinity chromatography and Western blot analysis with subunit-specific antibodies indicated that other endogenous subunits possibly present in these cells were not associated with beta 3 subunits. These results appear to indicate that most of the allosteric binding sites present on gamma-aminobutyric acid type A receptors can be formed by the beta subunit of these receptors. Homo-oligomeric beta 3 receptors therefore are an excellent model system for the structural investigation of gamma-aminobutyric acid type A receptors.
Slany
A
A
Department of Biochemical Psychiatry, University Clinic for Psychiatry, Vienna, Austria.
Zezula
J
J
Tretter
V
V
Sieghart
W
W
D000495:Allosteric Site; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000473:pathology* / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000502:physiology*; D013462:Sulfur Radioisotopes; D014162:Transfection
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018076:DNA, Complementary; D008024:Ligands; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D013462:Sulfur Radioisotopes; C037476:tert-butylbicyclophosphorothionate