title: [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.
abstract: Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, TX 76107, USA.
authors.forename: Monica A
authors.initials: MA
authors.lastname: Yagle
authors.forename: Michael W
authors.initials: MW
authors.lastname: Martin
authors.forename: Christopher M
authors.initials: CM
authors.lastname: de Fiebre
authors.forename: NancyEllen C
authors.initials: NC
authors.lastname: de Fiebre
authors.forename: John A
authors.initials: JA
authors.lastname: Drewe
authors.forename: Glenn H
authors.initials: GH
authors.lastname: Dillon
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D011485:Protein Binding; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C091518:1-(4-ethynylphenyl)-4-propyl-2,6,7-trioxabicyclo(2.2.2)octane