Picrotoxin Binding Sites Research Articles

Engineering a Prokaryotic Cys-loop Receptor with a Third Functional Domain

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Neurosteroids and GABA-A Receptor Function

Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABAA-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABAA-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions.

Ginkgolide B and bilobalide block the pore of the 5-HT3 receptor at a location that overlaps the picrotoxin binding site

Extracts from the Ginkgo biloba tree are widely used as herbal medicines, and include bilobalide (BB) and ginkgolides A and B (GA and GB). Here we examine their effects on human 5-HT3A and 5-HT3AB receptors, and compare these to the effects of the structurally related compounds picrotin (PTN) and picrotoxinin (PXN), the two components of picrotoxin (PTX), a known channel blocker of 5-HT3, nACh and GABAA receptors. The compounds inhibited 5-HT-induced responses of 5-HT3 receptors expressed in Xenopus oocytes, with IC50 values of 470μM (BB), 730μM (GB), 470μM (PTN), 11μM (PXN) and >1mM (GA) in 5-HT3A receptors, and 3.1mM (BB), 3.9mM (GB), 2.7mM (PTN), 62 μM (PXN) and >1mM (GA) in 5-HT3AB receptors. Radioligand binding on receptors expressed in HEK 293 cells showed none of the compounds displaced the specific 5-HT3 receptor antagonist [3H]granisetron, confirming that they do not act at the agonist binding site. Inhibition by GB at 5-HT3A receptors is weakly use-dependent, and recovery is activity dependent, indicating channel block. To further probe their site of action at 5-HT3A receptors, BB and GB were applied alone or in combination with PXN, and the results fitted to a mathematical model; the data revealed partially overlapping sites of action. We conclude that BB and GB block the channel of the 5-HT3A receptor. Thus these compounds have comparable, although less potent, behaviour than at some other Cys-loop receptors, demonstrating their actions are conserved across the family.

Allopregnanolone (3α-Hydroxy-5α-pregnan-20-one) Derivatives with a Polar Chain in Position 16α: Synthesis and Activity

The lipophilic nature of allopregnanolone prevents its user-friendly application in human medicine. On inspiration by previously prepared allopregnanolone with a 16alpha-bound tetraethylammonium salt, an attempt was made to produce allopregnanolone analogues with polar groups introduced into position 16alpha with the goal of increasing water solubility, brain accessibility, and potency of neuroactive steroids. The Michael addition to derivatives of pregn-16-en-20-one was the key reaction step. The link between the steroid skeleton and the new side chain was either a methylene group (when diethyl malonate was added) or an oxygen atom (when a hydroxy derivative was added). [(35)S]TBPS displacement was used to evaluate the products. Several carbamates (but not their parent alcohols) displaced TBPS from the picrotoxin binding site on GABA(A) receptors. Although none of them was more potent than the above ammonium salt, which stimulated this study, their nonionic nature should not prevent their passage into the brain.

The Location of a Closed Channel Gate in the GABAA Receptor Channel

Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-Å resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1–M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of α1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location.

Preserved Pharmacological Activity of Hepatocytes-Treated Extracts of Valerian and St. John's Wort

The two herbal extracts valerian (Valeriana officinalis L.) and St. John's wort (Hypericum perforatum L.) were studied for their metabolic changes upon incubation with freshly prepared rat hepatocytes and subsequently analysed phytochemically as well as pharmacologically in vitro. Quantitative HPLC analysis of valerian extracts revealed considerable metabolic activities with regard to sesquiterpenes and iridoids. The amount of acetoxyvalerenic acid decreased 9-fold, while that of hydroxyvalerenic acid correspondingly increased 9-fold due to O-deacetylation. The valepotriates didrovaltrate, isovaltrate and valtrate decreased 2-, 18- and 16-fold, respectively. However, the binding affinities of the incubated extracts to the benzodiazepine and picrotoxin binding site of the GABA (A) receptor were quite similar to those of the non-incubated extracts. Neither valerenic acids nor valepotriates exhibited any significant effect on the two binding sites when tested as single compounds. Therefore, either other constituents represent the active ones or multiple compounds are necessary for the observed inhibitory and allosteric effects at the GABA (A) receptor. Extracts of St. John's wort were less potently metabolised than valerian. The amount of pseudohypericin and the main flavonoids (hyperoside, rutin, isoquercitrin, quercitrin, quercetin and I3,II8-biapigenin) slightly decreased during the 4-h incubation period. Both the antagonist effect at the corticotropin-releasing factor (CRF) type 1 receptor and the binding inhibition at the 5-HT transporter were attenuated during the metabolic treatment. The reduced antagonist effect correlates with the decreasing amount of pseudohypericin known to be a CRF (1) receptor antagonist. In conclusion, the incubation of plant extracts with freshly prepared rat hepatocytes represents a useful approach to study the pharmacological action of metabolised plant extracts. The consistent pharmacological activity of both valerian and St. John's wort is concordant with the known clinical efficacy of pharmacological activities.

Picrotoxin Inhibition Mechanism of a γ-Aminobutyric AcidA Receptor Investigated by a Laser-Pulse Photolysis Technique

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.

Picrotoxin Blockade of Invertebrate Glutamate‐Gated Chloride Channels: Subunit Dependence and Evidence for Binding Within the Pore

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Structure-activity studies of fluoroaljyl-substituted γ-butyrolactone and γ-thiobutyrolactone modulators of GABA A receptor function

Dihydro-2(3 H)-furanones (γ-butyrolactones) and dihydro-2(3 H)-thiophenones (γ-thiobutyrolactones) containing fluoroalkyl groups at positions C-3, C-4, and C-5 of the heterocyclic rings were prepared. The anticonvulsant/convulsant activities of the compounds were evaluated in mice. Brain concentrations of the compounds were determined and the effects of the compounds on [ 35S]- tert-butylbicyclophosphorothionate ([ 35S]TBPS) binding to the picrotoxin site on GABA A receptors were investigated. The effects of the compounds on GABA A receptor function were studied using electrophysiological methods and cultured rat hippocampal neurons. Fluorination at C-3 results in either subtle or pronounced effects on the pharmacological activity of the compounds. When hydrogen are replaced with fluorines at the methylene carbon of an ethyl groups, as in 3-(1,1-difluoroethyl)dihydro-3-methyl-2(3 H)-furanone ( 1), the anticonvulsant actions of the compound are not much changed from those found for the corresponding alkyl-substituted analogue. In marked contrast, fluorination and the methyl carbon of the ethyl group, as in dihydro-3-methyl-3-(2,2,2-trifluoroethyl)-2(3 H)-furanone ( 3), produces a compound having convulsant activity. This convulsant activity seems to be due to an increased affinity of the compound for the picrotoxin site on GABA A receptors caused by an interaction that involves the trifluoromethyl group. Results obtained with γ-butyrolactones containing either a 3-(1-trifluoromethyl)ethyl or a 3-(1-methyl-1-trifluoromethyl)ethyl substitutent indicate that the interactions of the trifluoromethyl group with the picrotoxin binding site are subject to both stereochemical and steric constraints. Sulfur for oxygen heteroatom substitution, as in the corresponding γ-thiobutyrolactones, affects the type (competitive, non-competitive, etc.) of binding interactions that these compounds have with the picrotoxin site in a complex manner. Fluorination of alkyl groups at the C-4 and C-5 positions of γ-butyrolactones having convulsant activity increases convulsant potency.

Use-dependent and Use-independent blocking actions of picrotoxin and zinc at the GABA C receptor in retinal horizontal cells

The inhibitory actions of picrotoxin (PTX) and zinc on the GABA receptor in acutely isolated catfish cone horizontal cells were studied and compared using the whole-cell patch clamp technique. PTX blocked the GABAcurrent elicited by 30 μM GABA with IC 50 = 0.64 μM. Over a PTX concentration range of 1–100 μ,M, simultaneous application of PTX with GABA (30 uM) produced current transients at both the onset and offset of the drug pulse. When the PTX concentration was maintained before, during, and after GABA application, the current transients at the onset and offset of GABA application disappeared. Thus, these transients seem to reflect a slower initial action of PTX at, and faster washout of PTX from, the GABA receptor than GABA when they were co-applied. The full recovery from PTX inhibition required a second GABA application. Recovery could not be achieved by a prolonged wash in the absence of GABA. These results suggest that PTX effect its use-dependent. Zinc also potently blocked the GABA current elicited by 30 μM GABA with an IC 50 about an order of magnitude higher than that of PTX (IC 50 = 8.2 uM). However, only the onset, but not the offset current transient was observed when zinc was simultaneously applied with GABA. The full recovery of the GABA current from zinc inhibition was obtained after washing for 20 sec and did not require a subsequent GABA application. This indicates that the zinc effect is use-independent. Our findings suggest that: (1) the zinc binding site is on the surface of the GABA receptor molecule; (2) there is a PTX binding site that is probably inside the receptor and its access requires GABA binding to the receptor.

GABAA receptor binding in the aging rat inferior colliculus.

The inhibitory neurotransmitter GABA has been shown to be critically involved in shaping neuronal responses to simple and complex acoustic stimuli in the inferior colliculus. Studies in the rat and human inferior colliculus have suggested significant changes in functions related to GABA neurotransmission occur in the aged. These changes include significant decreases in GABA content, GABA release, GABA neurons, glutamate decarboxylase enzymatic activity, and GABAB receptor binding. Such changes within the inferior colliculus may affect the ability of elderly listeners to process complex acoustic signals, particularly in the presence of background noise. The present study was designed to examine the regional distribution and effects of aging on GABAA receptor binding sites in the Fischer 344 rat inferior colliculus using in vitro quantitative receptor autoradiography. [3H]GABA binding to GABAA receptors was significantly reduced in the inferior colliculus of young adult (3 months) and aged (18-26 months) rats when compared to 2-month animals. However, no significant changes were observed after 3 months of age. Single concentrations of tritiated GABAA receptor ligands (muscimol, t-butylbicycloorthobenzoate, and flunitrazepam) revealed no significant age-related changes in receptor binding in the inferior colliculus between 3 and 26 months of age. To characterize further the pharmacology of the GABAA receptor in the inferior colliculus, GABA modulation of the picrotoxin binding site was examined using [3H]t-butylbicycloorthobenzoate. When increasing concentrations of GABA were added to the incubation buffer, a significant decrease in binding was observed in the inferior colliculus of rats in each age group. In aged rats, the dose-response curve was shifted to the left, indicating an increase in the potency of GABA to inhibit [3H]t-butylbicycloorthobenzoate binding. Although no changes in GABAA receptor binding were detected in the inferior colliculus after 3 months of age, a significant alteration in interaction between the GABA and picrotoxin binding sites was observed in the inferior colliculus of aged rats when compared to 3-month-old young adults. This difference appears to reflect an increased sensitivity of the receptor to GABA modulation in aged rats and, thus, may serve as a compensatory mechanism to enhance GABAA receptor function in response to a presynaptic loss of inhibition.

Quantitative autoradiography of [formula omitted] binding to the GABA A receptor complex

4′- Ethynyl-4-n-[2,3- 3 H 2] propylbicycloorthobenzoate ([ 3H]EBOB) binding to the GABA A receptor complex was characterized autoradiographically in rat brain and then its binding in human brain was investigated. [ 3H]EBOB binding was saturable, specific and identified a single population of binding sites. The K d obtained from saturation was 4.59 nM. Picrotoxin produced dose-dependent inhibition of [ 3H]EBOB binding and saturation analysis indicated a competetitive interaction. Isoguvacine inhibited [ 3H]EBOB binding with regionally different effects. Bicuculline increased [ 3H]EBOB binding only in the cerebellar granule cell layer. In human cerebellum, a high level of [ 3H]EBOB binding sites was seen in the granule cell layer. These results suggest that [ 3H]EBOB binds to the picrotoxin binding site associated with the GABA A receptor complex, that regional differences in GABA A agonist and antagonist modulation of [ 3H]EBOB binding reflect underlying regional differences in GABA A receptor subunit composition, and that there is a species difference in GABA A receptor distribution between human and rat cerebellum.

Alpha-spirocyclopentyl- and alpha-spirocyclopropyl-gamma-butyrolactones: conformationally constrained derivatives of anticonvulsant and convulsant alpha,alpha-disubstituted gamma-butyrolactones.

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

Studies on picrotoxin binding sites of GABA A receptors in rat cortical synaptoneurosomes

Experiments were performed to characterize [ 35S]TBPS binding in rat cortical Synaptoneurosomes, which have vesicular structures containing both pre- and postsynaptic elements. Scatchard analysis revealed a single component of [ 35S]TBPS binding sites with K D and B max values of 76.1 nM and 1.97 pmoles/mg protein, respectively, under physiological conditions. GABA and muscimol inhibited [ 35S]TBPS binding in a concentration-dependent manner. IC 50 values of these GABA A agonists in displacing synaptoneurosomal [ 35S]TBPS binding are comparable to previously reported EC 50 values of the agonist-stimulated 36C1 − uptake in Synaptoneurosomes by these agents. Furthermore, the IC 50 values of these GABA A agonists were better correspondence to those determined by [ 3H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculline increased [ 35S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [ 35S]TBPS binding. Benzodiazepine agonists and inverse agonists (0.1 to 10 μM) did not show any effects on the binding in the absence of muscimol. However, benzodiazepine agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of synaptoneurosomal [ 35S]TBPS binding. In addition, an anesthetic steroid, THDOC, and pentobarbital inhibited synaptoneurosomal [ 35S]TBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [ 35S]TBPS binding by various ligands which interact with GABA A supramolecular complexes remain intact in Synaptoneurosomes. It appears that this preparation is useful for investigating correlation between functional 36Cl − uptake and individual binding studies of each of the GABA A receptor complex.

Interaction of La 3+ with GABA A receptors in rat cerebrocortical membranes as detected with [ 35S]t-butylbicyclophosphorothionate binding

Lanthanides at micromolar concentrations stimulated binding of [ 35S]t-butylbicyclophosphorothionate (TBPS), a highly sensitive marker for allosteric modulation of the chloride channel of GABA A receptors, to purified rat cerebrocortical synaptosomal membranes in the absence of GABA. This trivalent cation effect appeared to reflect a specific and direct interaction with GABA A receptors, and could not be mimicked by divalent metal ions including Ca 2+, Cd 2+, Co 2+, Sr 2+, Mg 2+ and Mn 2+. On the other hand, Zn 2+, a known allosteric regulator of GABA A receptors, inhibited TBPS binding, but its presence could not prevent stimulation of TBPS binding by La 3+. We further examined the lanthanide binding site by monitoring La 3+-induced changes in TBPS binding. La 3+ reduced the K d for TBPS without affecting B max. Stimulation of TBPS by La 3+ was not additive with the increased TBPS binding induced by other agonists, including GABA (at nanomolar concentrations), neurosteroids or benzodiazepines (in the absence of GABA). GABA at micromolar concentrations inhibited TBPS binding and its inhibitory effect was potentiated by neurosteroids and benzodiazepines, but not by La 3+. Thus, GABA, in the presence of La 3+, inhibited TBPS binding in a monophasic manner with a Hill coefficient of approximately 2 and an IC 50 of 6 μM. In the absence of La 3+, the dose-response profile for GABA became biphasic with an apperance of a stimulatory phase at concentrations of GABA lower than 1 μM. Analysis of the initial stimulation yielded a Hill coefficient of 1 and an EC 50 of 88 nM. These data are consistent with the stimulation of TBPS binding by GABA resulting from the occupation of its high affinity site and its inhibition of TBPS from binding occupation of a low affinity site. It appears that GABA binding at the high affinity site induced the conformational change(s) at the TBPS binding site (which is presumably located near the mouth of the chloride channel as the picrotoxin binding site), which is qualitatively similar to the conformational change induced by La 3+, neurosteroids or benzodiazepines (in the absence of GABA). These similar and nonadditive effects on TBS binding may reflect changes on the multimeric GABA A receptors prior to opening of the chloride channels. Finally, the lanthanide site on GABA A receptors examined here with La 3+ appears to be different from the Zn 2+ site, and from the previously reported monovalent and divalent cation recognition sites (Squires and Saederup, 1982, Mol. Pharmacol. 22, 327).

Involvement of gamma-aminobutyric acid and N-methyl-D-aspartate receptors in the inhibitory effect of ethanol on pentylenetetrazole-induced c-fos expression in rat brain.

The expression of c-fos mRNA in rat brain was induced by intraperitoneal administration of pentylenetetrazole (PTZ) and picrotoxin, which act on the picrotoxin binding site of the gamma-aminobutyric acid-benzodiazepine (GABA-BZ) receptor complex, by N-methyl-D-aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of PTZ and picrotoxin but not that of NMDA were blocked by ethanol and the NMDA-receptor antagonist, MK-801. Ro 15-4513 partially reversed the inhibitory effect of ethanol on PTZ-induced c-fos mRNA synthesis. Acute ethanol administration blocked the actions of PTZ and NMDA without affecting the response to kainic acid or caffeine. Taken together, these data suggest that ethanol blocks c-fos gene activation by noncompetitive antagonists of the GABA-BZ receptor via actions on both the NMDA and GABA receptors.

Aging related alterations in GABA A receptor subunit mRNA levels in Fischer rats

The influence of aging on the binding of ligands to picrotoxin binding sites as well as steady state levels of mRNA for various α subunits of γ-aminobutyric acid (GABA) receptor complex was investigated in male Fischer F-344 rats. In aged rats, the binding of [ 35 S]t- butyl-bicyclophosphorothionate (TBPS) was significantly reduced. This decrease in TBPS binding derived from a reduced density of binding sites, rather than from affinity changes, in both cerebral cortex and cerebellum. In aged rats, α 1 mRNA level decreased approximately 70% between age 6 months and 24 months in the cerebral cortex ( P < 0.005). In contrast, α 1 mRNA remained unchanged in the cerebellum of old rats. The association of a decrease in picrotoxin binding sites in the cerebral cortex with a decline in α 1 mRNA level in the cerebral cortex and in α 2 mRNA level in the cerebellum is indicated. α 6 mRNa level increased with age in the cerebellum. These findings indicate a selective age related modulation in the stoichiometry of GABA A receptor in aging.

Aging reduces the mRNA of α1 GABA A receptor subunit in rat cerebral cortex

The effect of aging on the binding of ligands to the GABA, benzodiazepine and picrotoxin binding sites as well as α subunit mRNA level of GABA A receptor was investigated in cerebral cortex of male Fischer F-344 rats. In aged (730- to 770-day-old) rats, the binding of [ 35S]t-butylbicyclophosphorothionate (TBPS) was significantly reduced. Also, α 1 mRNA level was markedly decreased (86% suppression). In contrast, α 1 mRNA remained unchanged in cerebellum. These findings indicate a selective age-related structural change in GABA A receptor in rat cerebral cortex.

Development of Benzodiazepine and Picrotoxin (t‐Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.

Evidence that clomethiazole interacts with the macromolecular GABA A-receptor complex in the central nervous system and in the anterior pituitary gland

Clomethiazole (CLOM) is known to be an anticonvulsant drug and has been also reported to decrease serum prolactin (PRL) in humans. Both effects may be mediated by an enhancement of gabaergic transmission. In order to determine if (CLOM) interacts with GABA metabolism and/or at the GABA receptor level, we studied its effect on PRL release and on the binding of various compounds that interact with the GABAA-benzodiazepine-receptor complex. Intraperitoneal (IP) administration of CLOM to rats significantly decreased PRL levels, and this effect was antagonized by IP administration of bicuculline, an antagonist of the GABAA receptor. In vitro, the inhibitory effect of muscimol on PRL release from rat hemiadenohypophysis was potentiated in a dose-dependent manner by preincubation with CLOM. This effect was antagonized by picrotoxin (10(-6) M). On the other hand, CLOM had no effect on GABA metabolism and did not compete with GABAA, GABAB or benzodiazepine binding sites in cortical membranes. CLOM competed, however, with the picrotoxin binding site labelled with [35S]-butylbicyclophosphorothionate (TBPS), at an IC50 value of 1.2 x 10(-4) M, which is in the same range as some barbiturates. These results concerning PRL release and binding experiments with cortical membranes suggest that CLOM interacts with the picrotoxin/barbiturate site of the GABAA-receptor-chloride channel complex.