TBPS Binding Sites Research Articles

Autoradiographic study of neurosteroid binding sites labelled with [ 35S]-TBPS in brain of different species

Distribution of [ 35S]-TBPS binding sites was studied in various structures of brain in mouse and guinea pig and in cortex of monkey and in hippocampus of postmortem human brain. As it is observed for rat brain, high densities of [ 35S]-TBPS binding sites were found in layer IV of cortex in the four species, and in thalamus of mouse and guinea pig. Intermediate densities of binding sites were observed in superficial and deep layers of cortex in those four species and in hippocampus of mouse, guinea pig, and human. In all brain structures studied. 5α3αP and picrotoxin produced a dose-dependant inhibition of [ 35S]-TBPS binding. No significant interregion or interspecies differences could not be detected for IC 50 values of 5α3αP or picrotoxin to inhibit [ 35S]-TBPS from its binding sites. In all regions studied, IC 50 values were close to 1.5 × 10 −6 M for 5α3αP and 2.3 × 10 −7 M for picrotoxin.

Alterations in the gamma-aminobutyric acid-gated chloride channel following transient forebrain ischemia in the gerbil.

The role of inhibitory neurotransmission in selective neuronal degeneration after transient forebrain ischemia was studied by binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to the gamma-aminobutyric acid (GABA)-gated chloride channel and measurement of GABAA receptor function in Mongolian gerbil brain. [35S]TBPS binding to the hippocampus, striatum, and cortex quantified by autoradiography and muscimol-stimulated 36Cl- uptake in synaptoneurosomes of the same regions were examined 1, 4, and 29 days after a 5-min bilateral carotid occlusion. [35S]TBPS binding was decreased in the pyramidal cell dendritic layers, stratum oriens, and stratum lacunosum-moleculare of the CA1 hippocampus, 4 and 29 days after occlusion, and in the stratum radiatum 29 days after occlusion. [35S]TBPS binding sites in the lateral striatum decreased 47% 4 days after occlusion. At the same time, there was a corresponding decrease in muscimol-stimulated 36Cl- uptake in the striatal synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in the hippocampus decreased slightly 4 days after occlusion and more so after 29 days, although these decreases were not significant. No changes were observed in somatosensory cortex at any time point. These data suggest that a portion of GABAA receptors in areas sensitive to ischemic insult are associated with degenerating neurons, whereas other GABAA) receptors are spared.

Foot-shock stress enhances the increase of [35S]TBPS binding in the rat cerebral cortex and the convulsions induced by isoniazid

We report earlier that isoniazid and foot-shock stress individually increase the maximal number of [35S]TBPS binding sites (Bmax) measured "ex vivo" in unwashed membranes from rat cerebral cortex and that the increase due to both treatments are prevented by pretreatment "in vivo" with diazepam which alone induced a significant decrease in the total number of [35S]TBPS binding sites. In the present paper, the effect of stress was studied on both the increase in [35S]TBPS binding and the convulsant activity induced by isoniazid in unstressed rats. Isoniazid induced a time dependent increase in [35S]TBPS binding. The isoniazid-induced increase in [35S]TBPS binding was markedly potentiated by foot-shock stress. Moreover, foot-shock stress markedly reduced the latency to the appearance of generalized seizures induced by isoniazid (300 mg/kg s.c.). The results provide evidence that the "in vivo" inhibition of GABAergic transmission elicited by isoniazid results in an increase of [35S]TBPS binding in the rats cerebral cortex. The finding that stress, like isoniazid, enhances [35S]TBPS binding suggests that this treatment also inhibits the function of GABAergic synapses.

Inactivation of [ 35S]t-butylbicyclophosphorothionate binding sites by the arginine reagent, 2,3-butanedione

The “cage” convulsant [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) binds with high affinity to specific sites “at” or “near” a γ-amino-butyric acid (GABA)-gated chloride channel according to current hypothesis. We now report that pretreatment of membranes with 2,3-butanedione, an arginine-specific reagent, causes a dose- and time-dependent decrease in the number of [ 35S]TBPS binding sites. No decrease occurs when membranes are pretreated with 2,3-butanedione in the presence of picrotoxinin. Binding of [ 35S]TBPS to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

GABA receptors of insects susceptible and resistant to cyclodiene insecticides

AbstractToxicity tests revealed up to 40‐fold resistance to a number of cyclodiene insecticides in a laboratory‐reared, cyclodiene‐resistant (CYW) housefly strain (Musca domestica L.). Using [35S] TBPS as a probe for convulsant sites in insects, saturable specific binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of[35S] TBPS binding data to CSMA and CYW membranes failed to provide evidence for significant differences between the two strains in either the affinity (Kd) or density (Bmax) of saturable binding sites. For several polychlorocycloalkane insecticides, the ligand displacement profile of [35S] TBPS binding was almost identical for the CSMA and CYW houseflies. Therefore, using [35S] TBPS as a probe for convulsant sites, a 40‐fold resistance to cyclodienes in the CYW housefly strain cannot be accounted for only in terms of alterations in TBPS binding sites.

Lindane inhibition of [ 35S]TBPS binding to the GABA A receptor in rat brain

The inhibition of [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) binding to the GABA A receptor by the insecticide γ-hexachlorocyclohexane, lindane, was studied in several brain regions and using different membrane preparation methods, both in vitro and after dosing the animals with the chemical. In the latter studies, the amount of lindane remaining in the membrane suspensions used for binding assays was determined. In vitro data showed values of IC 50 from 150 to 1675 nM, varying in function of the membrane preparation method used. This may account for the discrepancies in IC 50 values found in the literature. IC 50 values within the range of 150–250 nM were determined using extensively washed membranes from several brain regions, so no evidence arose for brain regional differences in the affinity of lindane for the TBPS binding site. After different schedules of acute treatment with lindane, we found a manifest relationship between the extent of the observable inhibition of [ 35S]TBPS binding and the lindane amount remaining in the membrane suspensions used for binding assays. This relationship was in good agreement with the in vitro data, so no support for an in vivo acute regulation of the binding site was obtained.

Decrease in the Function of the γ‐Aminobutyric Acid‐Coupled Chloride Channel Produced by the Repeated Administration of Pentylenetetrazol to Rats

The acute administration of pentylenetetrazol (PTZ; 25-75 mg/kg i.p.) failed to modify the specific binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [35S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of gamma-aminobutyric acid (GABA)-stimulated 36Cl- uptake was measured in the cerebral cortex of PTZ-treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA-coupled chloride channel activity that may be related to the chemical kindling produced by this compound.

Blocking actions of heptachlor at an insect central nervous system GABA receptor

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range -75 mV to -110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate [( 35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.

35S]TBPS binding sites are decreased in the colliculi of mice with a genetic predisposition to bicuculline-induced seizures

Several differences have been found in GABAergic function between the long sleep (LS) and short sleep (SS) mice which were genetically selected for different ethanol-induced sleeptimes, and it has been suggested that these differences may explain their differential ethanol sensitivity. However, these lines also differ in seizure susceptibility, a behavior which may also be mediated by GABAergic pathways. Thus, it is difficult to associate differences in GABA neurochemistry with either of these behaviors, particularly when only two selected lines are used. We measured differences in the density and affinity of the [ 35S]TBPS binding site on the GABA A receptor/Cl − ionophore complex in discrete brain areas; and in order to determine the relationship between receptor binding and behavioral differences, we included mice from 5 of the LS and SS recombinant inbred strains (LS × SS RI) in addition to mice from the LS and SS lines. [ 35S]TBPS binding in sagittal brain sections was analyzed by quantitative autoradiography, and the amount of binding differed depending on whether bicuculline was added to inhibit endogenous GABA binding. In the presence of bicuculline, the number of [ 35S]TBPS sites in SS mice was highest in the colliculi (4.5 ± 0.5 pmol/mg protein), cerebellum (4.8 ± 0.6 pmol/mg), hippocampus (3.2 ± 0.7 pmol/mg) and cortex (2.9 ± 0.3 pmol/mg). The B max was two-fold lower in both superior and inferior colliculi (IC) of LS mice. There were no differences between lines in B max in any other area and in K d values in any area (58 ± 4.0 nM). When we compared [ 35S]TBPS binding in the LS × SS RI strains, B max values in IC were negatively correlated with latency to bicuculline-induced seizures but were not correlated with the duration of ethanol-induced sleeptime. These data support the hypothesis that differences in GABAergic neurochemistry in these mouse lines may not always be related to differences in ethanol sensitivity and point to GABAergic receptors in IC as possibly being involved in determining seizure sensitivity to bicuculline.

Dramatic increase in nigral t-[ 35S]butylbicyclophosphorothionate binding sites elicited by the degeneration of the striato-nigral GABAergic pathway: reversal by diazepam

In rats, the degeneration of the striato-nigral GABAergic pathway caused by the intrastriatal injection of kainic acid induced a marked decrease (65%) of GABA content and glutamic acid decar☐ylase (GAD) activity and a dramatic increase (225%) in the binding of t-[ 35S]butylbicyclophosphorothionate ([ 35S]TBPS) to a membrane preparation from the substantia nigra homolateral to the injected striatum. The increase in [ 35S]TBPS binding in the denervated substantia nigra was exclusively due to an increased density of binding sites ( B max) with no change in the dissociation constant ( k d). The enhancement in [ 35S]TBPS binding was almost completely reversed by the intraperitoneal administration of diazepam (3 mg/kg) to kainic acid-lesioned rats. Moreover, diazepam produced a significant decrease (30%) in the density of [ 35S]TBPS binding sites also in the sham-operated side. In contrast the ‘in vitro’ addition of the GABA A receptor antagonist bicuculline (1 μM) to the membrane preparation from the denervated substantia nigra further increased [ 35S]TBPS binding. These findings suggest the view that the increase of nigral [ 35S]TBPS binding is directly related to the inhibition in the function of nigral GABAergic synapses following the loss of the striato-nigral GABAergic pathway. Our results indicate that [ 35S]TBPS binding to brain structure is a potential tool to reveal alteration in the function of GABA A receptor complex elicited by physiological, pharmacological and pathological conditions.

Differential Effects of Iodide and Chloride on Allosteric Interactions of the GABAA Receptor

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.

Isoniazid, an inhibitor of GABAergic transmission, enhances [ 35S]TBPS binding in rat cerebral cortex

Isoniazid, administered to rats one hour before killing produced a dose-dependent enhancement of [ 35S]t-butylbiciclophosphorothionate ([ 35S]TBPS, 2 nM) measured ex vivo in unwashed membrane preparation of the cerebral cortex. Saturation experiments revealed that the effect of isoniazid was due to an increase (+ 36%) in the total number of [ 35S]TBPS binding sites. Diazepam (3 mg/kg i.p.) administered 15 min after isoniazid the enhancement of [ 35S]TBPS binding elicited by isoniazid. Moreover, diazepam itself induced a significant decrease (−30%) in the total number of [ 35S]TBPS binding sites. These results provide the first direct evidence that ‘in vivo’ alterations in the function of the GABA-dependent chloride channel can be detected in vitro by studying the binding of [ 35S]TBPS to its recognition sites in the GABA A receptor complex. Our finding suggests a new model suitable to study biochemically the function of GABAergic synapses under various physiological and pharmacological conditions.

Binding characteristics of t-[35S]butylbicyclophosphorothionate in discrete brain regions of rats made tolerant to and dependent on pentobarbital.

The effects of acute and continuous pentobarbital administration by pellet implantation on binding characteristics of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) in discrete regions of rat brains were examined. Acute administration of pentobarbital (60 mg/kg, s.c.) affected neither the KD nor the Bmax values of [35S]TBPS binding in any of the regions studied. The cerebella of pentobarbital-tolerant rats had an increased density of [35S]TBPS binding sites with no change in their apparent affinity. There were no significant changes in the binding characteristics in the frontal cortex (FC), the striatum (ST), and the substantia nigra (SN) of these animals. Twenty-four hours after removal of the pentobarbital pellets, a significant decrease in the latency of onset of first twitch response induced by pentylenetetrazol (PTZ) (50 mg/kg, i.p.) was observed. In addition, the density of [35S]TBPS binding sites was significantly increased in the FC, the SN, and the cerebellum but not in the ST. In all brain regions studied, placebo pellet implantation and pentobarbital tolerance and dependence caused no changes in the apparent affinity of [35S]TBPS binding or the IC50 of pentobarbital for the inhibition of [35S]TBPS binding. These results suggest that [35S]TBPS binding was significantly increased following the withdrawal of the pentobarbital pellets without altering intrinsic coupling activity of barbiturate recognition sites and convulsant binding sites and that these increases in [35S]TBPS binding are related to the increased susceptibility to seizures induced by PTZ in rats made dependent on pentobarbital.

Effects of Picrotoxin Treatment on GABAA Receptor Supramolecular Complexes in Rat Brain

The effects of acute and chronic administration of a subconvulsive dose of picrotoxin on t-[35S]butylbicyclophosphorothionate ([3S]TBPS), [3H]muscimol, and [3H]flunitrazepam binding characteristics in various regions and on the convulsant potency of picrotoxin in Sprague-Dawley rats were examined. Acute administration of a subconvulsive dose of picrotoxin (3 mg/kg, i.p.) significantly increased [35S]TBPS and [3H]muscimol binding in cerebellum (CB) with no change in frontal cortex (FC). In rats treated chronically with picrotoxin (3 mg/kg, i.p., daily for 10 days), the Bmax of [35S]TBPS binding site was significantly decreased in the FC, striatum (ST), and CB with no change in KD values. Neither [3H]muscimol binding in the FC and CB nor [3H]flunitrazepam binding in the FC was affected in these rats. In addition, the potency of pentobarbital to inhibit [35S]TBPS binding in vitro was not altered following acute or chronic treatment of picrotoxin. Chronic administration of picrotoxin did not affect convulsive ED50 or LD50 of picrotoxin; however, it delayed the onset of convulsions and increased the time to death. These results suggest that treatment with picrotoxin at a subconvulsive dose for 10 days causes down-regulation of [35S]TBPS binding sites and that this down-regulation might be related, at least in part, to the decreased extent of convulsant potency of picrotoxin. In addition, the results indicate possible interaction between convulsant binding sites and GABAA receptor sites in the CB following picrotoxin treatment.

Effect of anticonvulsant drugs on (35S)t-butylbicyclophosphorothionate binding in vitro and ex vivo.

Using several concentrations of eight anticonvulsant drugs in clinical use (carbamazepine, clonazepam, phenytoin, phenobarbital, ethosuximide, primidone, sodium valproate, and D,L-gamma-vinyl GABA), we studied their abilities in vitro to displace (35S)t-butylbicyclophosphorothionate (35S-TBPS) from its binding site in a homogenate of rat brain. Thereafter ethosuximide (150 mg/kg), phenobarbital (30 mg/kg), clonazepam (0.3 mg/kg), or phenytoin (100 mg/kg) was injected intraperitoneally into rats for 16-20 days; and the effect of drug administration on 35S-TBPS binding was studied in the cortex and hippocampus ex vivo. Phenobarbital (100 microM, P less than 0.001), ethosuximide (500 microM, P less than 0.001), and phenytoin (40 microM, P less than 0.001) decreased the specific 35S-TBPS binding in vitro by 10-16%. After drug administration of phenobarbital (concentration in plasma 168 microM), the number of binding sites decreased and the binding affinity (P less than 0.05) in the cortex increased. Other anticonvulsants did not modulate 35S-TBPS binding in vitro at the concentration analogous to therapeutic plasma levels or ex vivo at the dose used. These results suggest that the use of phenobarbital may modulate the TBPS binding site, but the role of the present findings in the anticonvulsant action of phenobarbital needs to be further studied.

GABA A receptor blockers reverse the inhibitory effect of GABA on brain-specific [ 35S]TBPS binding

Thirteen sustances previously reported to antagonize the electrophysiological effects of γ-aminobutyric acid (GABA) on neurons also reversed the inhibitory effects of GABA on specific [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) binding to sites on rat brain membranes in vitro with a rank-order of potentcies similar to those found in electrophysiological (R 5135 > pitrazepin > bicuculline> SR 95103 > securinine) confirming the earlier conclusion that GABA inhibits [ 35S]TBPS binding by acting allosterically on relevant GABA A receptors. Pitrazepin is the most potent of a series of mono N-aryl piperazines that block GABA A receptors. The new aryl amino pyridazine GABA derivative SR 95531 was about 3-fold more potent than bicuculline and 39-fold more potent than the structurally related SR 95103. Four known GABA antagonists have the same rank orders of potencies as convulsants and as reversers of GABA's inhibitory action on [ 35S]TBPS binding (bicuculline > securinine > theophylline > caffeine). Reversal of GABA-induced suppression of [ 35S]TBPS binding provides a simple method for further characterizing GABA A receptors linked to TBPS binding sites, and facilitates identification of convulsants and novel, perhaps selective, GABA antagonists.

The GABAa receptor complex in the developing chick optic tectum: Ontogeny of [3H]muscimol, [3H]flunitrazepam and [35S]TBPS binding sites

The developmental profiles of the neurotransmitter recognition site, labeled by [(3)H]muscimol, and of the two main modulatory sites, labeled by [(3)H]flunitrazepam and [ (35)S ]t- butylbicyclophosphorothionate , respectively, within the GABA(A) receptor complex, have been determined in chick tectal lobes between embryonic day 8 and postnatal day 20. The consonance among the rates of appearance and accumulation of the three receptor sites in tectal membranes suggests a coordinated expression and assembly of the protein subunits involved in the spatial configuration of the complex and its three binding sites, although the existence of isolated muscimol binding subunits during early embryogenesis cannot be excluded at the present time. Furthermore, the total number of binding sites of each kind, per pair of lobes, is compatible with a 1/1/I stoichiometry. The GABA(A) receptor complex reaches a maximum of expression, relative to total membrane protein, immediately after hatching, suggesting that the tectal GABAergic system may be instrumental in damping the effects of sudden exposure to light of the chick visual system upon eye opening.

Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore

The toxicity to mice of intraperitoneally-administered polychlorocycloalkane (PCCA) insecticides is generally correlated with their potency as in vitro inhibitors of the brain specific [ 35S]- t -butylbicyclophosphorothionate ([ 3S]TBPS) binding site with correction for metabolic activation and detoxification. These findings from our earlier studies are extended here to in vivo investigations relating convulsant action to inhibition of the TBPS binding site in poisoned mice. Radioligand binding assays involved brain P 2 membranes washed three times with 1 mM EDTA to remove endogenous γ-aminobutyric acid (GABA) or other modulator(s) which otherwise serves as a noncompetitive inhibitor of [ 35S]-TBPS binding at the GABA-regulated chloride ionophore. Examination of lindane, technical toxaphene, toxaphene toxicant A, and 10 polychlorocyclodiene insecticides revealed 62 ± 4% binding site inhibition 30 min after their LD 50 doses with 32 ± 3% inhibition at one-half and 6 ± 3% inhibition at one-quarter of their LD 50 doses. This correlation between binding site inhibition and convulsant action is also evident in dose- and time-dependency studies with endosulfan sulfate. The brain P 2 membranes of treated mice contain the parent compound with each of the PCCAs plus activation products of some of the cyclodienes, i.e. endosulfan sulfate from α- and ß-endosulfan and 12-ketoendrin from isodrin and endrin. The finding that the brains of treated mice contain sufficient PCCA or its activation products to achieve a magnitude of [ 35S]TBPS binding site inhibition correlated with the severity of the poisoning signs supports the hypothesis that the acute toxicity of PCCA insecticides to mammals is due to disruption of the GABA-regulated chloride ionophore.

Effects of insecticides and GABAergic agents on a house fly [ 35S] t-butylbicyclophosphorothionate binding site

Specific binding of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) to a house fly thorax-plus-abdomen membrane preparation at 20°C is characterized by apparent K d and B max values of 0.21 μ M and 2.5 pmol/mg protein, respectively, an association half-time of 13 min at 2 n M, and a biphasic dissociation curve showing half-times of 15 and 35 min. Specific binding is reduced at 37°C apparently due to instability of the receptor-ligand complex and at 0°C as the result of very slow association. [ 35S]TBPS binding is diminished by detergents, stimulated by GABA at low ligand concentration, and inhibited by picrotoxinin and certain barbiturates, benzodiazepines, bicyclophosphorus compounds, and polychlorocycloalkane insecticides. The potency of TBPS and three related phosphorothionates in displacing [ 35S]TBPS parallels their toxicity on injection into house flies; the corresponding bicyclophosphates are less active in both assays. Cyclodienes of low toxicity are generally poor inhibitors of radioligand binding. α-Endosulfan and syn-12-hydroxyendrin are more potent than their β and anti isomers, respectively, both as inhibitors of TBPS binding and as toxicants. Analysis of Scatchard plots indicates that picrotoxinin and heptachlor epoxide are non-competitive inhibitors of [ 35S]TBPS binding. The [ 35S]TBPS binding site of the house fly membrane preparation differs from that extensively studied in mammalian brain with respect to their responses to many insecticides and GABAergic agents.

GABA, depressants and chloride ions affect the rate of dissociation of 35S-t-butylbicyclophosphorothionate binding

The dissociation of 35S-TBPS was studied from binding sites of rat cerebral cortex. Monophasic dissociation plots became polyphasic and accelerated in the presence of micromolar concentrations of GABA suggesting the involvement of low (or super-low) affinity GABA receptors. The presence of the depressants etazolate, R(-)MPPB and ethanol resulted in similarly accelerated dissociation patterns. In contrast, the convulsants S(+)MPPB and pentamethylenetetrazol did not significantly affect the dissociation of TBPS. Dissociation initiated by dilution was not affected either by an excess of picrotoxin or by varying the equilibrium occupancy of the TBPS sites. These findings rule out the possibility of a kinetic cooperativity for the binding of convulsants. The removal of chloride ions also enhanced the rate of TBPS dissociation. Kinetic heterogeneity of the TBPS binding sites can be interpreted with allosteric interactions mediated by various sites at the GABA receptor complex coupled to different states of the chloride ionophore.