[formula omitted]]Ethynylbicycloorthobenzoate ([ [formula omitted]]EBOB) Binding in Recombinant GABA A Receptors

Abstract

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABA A 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 GABA A receptor. We have thus examined [ 3H ]EBOB binding in HEK293 cells stably expressing human α1β2γ2 and α2β2γ2 GABA A 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 α1β2γ2 and α2β2γ2 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 μM 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 μM) inhibited [ 3H ]EBOB binding. Overall, our data demonstrate that [ 3H ]EBOB binds to a single, high affinity site in α1β2γ2 and α2β2γ2 GABA A 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 GABA A receptor.