Dissociation of [³⁵S]t‐Butylbicyclophosphorothionate Binding Differentiates Convulsant and Depressant Drugs that Modulate GABAergic Transmission

Abstract

The dissociation of [35S]t-butylbicyclophosphorothionate ([35S]TBPT) from binding sites on membranes from rat cerebral cortex, after addition of saturating concentrations of convulsant and depressant drugs, was studied. The addition of unlabeled TBPT, picrotoxinin, or pentamethylenetetrazol resulted in dissociation patterns that were monophasic and not distinguishable, suggesting that these convulsants bind competitively to the same (convulsant) sites. In contrast, gamma-aminobutyric acid (GABA) greatly facilitated [35S]TBPT dissociation by binding allosterically to the GABA recognition site of the receptor-ionophore complex. TBPT dissociation was similarly accelerated by the depressants etazolate, (+)-etomidate, and barbiturates. The convulsant and depressant S(+) and R(-) stereoisomers of N-methyl-5-phenyl-5-propyl-barbituric acid displayed large stereoselectivity in the acceleration of TBPT dissociation. These results suggest that depressants bind to sites different from the convulsant sites of the allosteric GABA receptor complex, or the binding of depressants to the same population of sites elicits negative cooperativity and dissociates the convulsants.

A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.

“…The resistance ratio for picrotoxin derived from animal toxicity assays (2 x 105) differed widely from the present value of 116 based on measurements at the receptor level. This probably reflects both metabolism of picrotoxin and a non-linear relation between the insecticide concentration and uptake in contact assays of resistance in insects (Bloomquist et al 1991; (Maksay & Ticku, 1985). Although cyclodiene-resistant house flies exhibit a significant resistance to TBPS, the relevance of this ligand to the cyclodiene binding site is equivocal (Anthony, Benner, Rauh & Sattelle, 1992).…”

“…The classical GABAA receptor antagonist picrotoxin competes with cyclodienes in binding assays (Sattelle, 1990). The caged convulsant t-butyl-bicyclophosphorothionate (TBPS) displaces picrotoxin (Maksay & Ticku, 1985). Thus, a number of ligands interact with this region of the receptor.…”

Receptor Subtype-Dependent Positive and Negative Modulation of GABAA Receptor Function by Niflumic Acid, a Nonsteroidal Anti-Inflammatory Drug

“…The GABA A receptor convulsant [ 35 S]t-butylbicyclophosphorothionate (TBPS) specifically binds to a picrotoxininsensitive site associated with the GABA A receptor ionophore (Squires et al, 1983 (Squires et al, 1983;Maksay and Ticku, 1985), whereas negative modulators, such as bicuculline, block this effect of GABA (Squires This study was partially supported by funds from the Academy of Finland (to E.R.K. ).…”

“…35 S]TBPS binding (Squires et al, 1983;Maksay and Ticku, 1985), whereas negative modulators, such as bicuculline, block this effect of GABA (Squires and Saederup, 1987). Furosemide, presently the most subtype-selective antagonist, noncompetitively increases the binding in the cerebellar granule cell layer and ␣6 subunitcontaining recombinant receptors (Korpi et al, 1995 36 Cl Ϫ flux assay (Im and Blakeman, 1991).…”

“…GABA A ergic drugs, such as benzodiazepines, barbiturates, and volatile anesthetics, enhance the actions of GABA (Sieghart, 1995) and are used to treat, e.g., anxiety, insomnia, and epilepsy, and in general anesthesia. Many drug effects on GABA A receptor function have been shown to depend on subunit combinations (subtypes) and even on critical amino acids in specific subunits (Korpi et al, 2002).The GABA A receptor convulsant [ 35 S]t-butylbicyclophosphorothionate (TBPS) specifically binds to a picrotoxininsensitive site associated with the GABA A receptor ionophore (Squires et al, 1983 (Squires et al, 1983;Maksay and Ticku, 1985), whereas negative modulators, such as bicuculline, block this effect of GABA (Squires This study was partially supported by funds from the Academy of Finland (to E.R.K. ).…”