TBOB Binding Research Articles

ETHANOL-INDUCED ALTERATIONS IN THE FUNCTION OF CEREBRAL GABA <sub>A</sub> RECEPTOR COMPLEX: EFFECT ON GABA-DEPENDENT <sup>36</sup> Cl <sup>−</sup> INFLUX INTO CEREBRAL MEMBRANE VESICLES

Effect of addition in vitro of ethanol on the functions of the GABAA receptor complex was investigated using synaptic membrane preparation obtained from mouse brain. Ethanol (50-200 mM) had no significant effect on the specific bindings of [3H]muscimol to GABAA receptor and [3H]flunitrazepam to benzodiazepine receptor in cerebral particulate preparations. However, ethanol induced an inhibition of [3H]TBOB binding to Cl- channel and a suppression of flunitrazepam-induced enhancement of [3H]muscimol binding and of salsolinol-induced accentuation of [3H]flunitrazepam binding to cerebral particulate fraction. In contrast, ethanol facilitated GABA-dependent 36Cl- influx but eliminated the stimulatory effects of flunitrazepam and salsolinol on GABA-dependent 36Cl- influx into cerebral membrane vesicles. These results suggest that ethanol may facilitate the function of GABA-gated chloride channel in spite of inducing deteriorations of antagonist binding capacity of chloride channel as well as of the functional coupling between GABAA receptor and benzodiazepine receptor in the brain.

Binding of the cage convulsant, [ 3H]TBOB, to sites linked to the GABA A receptor complex

[ 3H]t-Butylbicycloorthobenzoate ([ 3H]TBOB) binds to specific sites on crude synaptic rat brain membranes. The dissociation constant, K d, determined from saturation experiments is near 8 nM and the receptor density B max is about 20 pmol/g wet tissue. Non-specific binding constitutes about 35% of the total binding at 4 nM [ 3H]TBOB. The association of [ 3H]TBOB is monophasic but its dissociation is biphasic. K d values of 8 nM (70% of the binding sites) and 20 nM (30% of the binding sites) were estimated from the kinetic data. These values differ from those previously reported. Specifically bound [ 3H]TBOB is displaced by picrotoxin and by t-butylbicyclophosphorothionate (TBPS). No simple competitive interaction of picrotoxin with [ 3H]TBOB binding was found. Micromolar quantities of the GABAergic facilitating compounds, GABA, muscimol and diazepam inhibited [ 3H]TBOB binding in an allosteric manner.

Ontogenesis and properties of the convulsant recognition site(s) of the γ-aminobutyric acid (GABA) receptor complex in chicken embryo

Ontogenesis of the convulsant (picrotoxinin or t-butylbicyclophosphorothionate (TBPS) site(s) was defined in chicken embryonic brain with the radioligands [ 35S]TBPS and [ 3H] t-butylbicycloorthobenzoate ([ 3H]TBOB). Binding of the radioligands is detectable from day 6 of incubation. The increase in binding after day 14 of incubation is due to an increase in the number of binding sites. The pharmacological properties of the embryonic recognition site(s) do not undergo significant changes during hatching based on the affinity of embryonic and chick brain membranes for [ 3H]TBOB; the rate of association and dissociation for TBOB; non-competitive inhibition of [ 3H]TBOB binding and modulation of 1R, αS-cis-cypermethrin interaction with the recognition site(s) by GABA; and inhibition of radioligand binding by endrin, picrotoxinin and TBPS. Early development and in vitro susceptibility of the recognition site to convulsive toxicants makes the embryo a possible target for a variety of drugs and environmental toxicants acting at this site.

The influence of folic acid on the picrotoxin-sensitive site of the GABA a-receptor complex

The site of action responsible for the convulsive effect of folic acid was investigated in vitro. Folic acid ( EC max 5 × 10 −4 M) enhances the binding of the cage convulsant [ 3H];-butylbicycioorthobenzoate ([ 3H]TBOB) to rat brain membranes, namely to 130% of control in the absence of GABA and to over 300% of control in the presence of physiological concentrations of GABA. Analysis of the binding parameters reveals that folic acid increases the apparent number of [ 3H]TBOB binding sites.