Biochemical pharmacology of the gamma-aminobutyric acid receptor/ionophore protein

The synaptic receptor sites for the neurotransmitter gamma-aminobutyric acid (GABA) can be assayed in vitro with several radiolabeled agonists and one antagonist. Numerous criteria of specificity have been met for these binding sites. All of the ligands show heterogeneity in binding affinities. The subpopulations thus defined have a remarkably similar specificity for GABA analogs, which suggests an intimate relationship and possible interconvertibility. Modulation of GABA receptor binding by barbiturates, anions, and other membrane treatments that affect agonists and antagonists in an opposite manner suggests a three-state model of interconvertible affinities. The complex of GABA receptor and chloride ion channel contains modulatory sites for barbiturates and benzodiazepines, drugs that enhance GABA responses in neurons. The receptor complex can be solubilized in detergent with the three mutually interacting receptor activities intact. The complex has an apparent molecular weight of 355,000 and has been partially purified. GABA agonist function has been assayed at the biochemical level by measuring the activation of 36Cl- efflux from preloaded hippocampal slices by GABA, muscimol, and barbiturates. This response is blocked by the antagonists of the GABA site (bicuculline) and the barbiturate site (picrotoxin). Comparison of binding and function on the same tissue should be useful in analyzing the mechanism of action of GABA.     

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.     

The GABA postsynaptic membrane receptor-ionophore complex

Summary The function of the inhibitory neurotransmitter, -aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered GABA-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developedin vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.     

Effect of ergot alkaloids on 3H-flunitrazepam binding to mouse brain GABAA receptors

In vitro effects of dihydroergotoxine, dihydroergosine, dihydroergotamine, alpha-dihydroergocriptine (ergot alkaloids), diazepam, methyl-beta-Carboline-3-carboxilate (beta-CCM), flumazenil (benzodiazepines), gamma-amino butyric acid (GABA) and thiopental (barbiturate) were studied on mouse brain (cerebrum minus cerebral cortex) benzodiazepine binding sites labeled with 3H-flunitrazepam. Specific, high affinity (affinity constant, Kd = 57.7 8.6 nM) binding sites for 3H-flunitrazepam on mouse brain membranes were identified. All benzodiazepine drugs inhibited 3H-flunitrazepam binding with nanomolar potencies. In contrast to benzodiazepines, all ergot drugs, GABA and thiopental produced an enhancement of 3H-flunitrazepam binding to its binding site at the GABAA receptor of the mouse brain. The rank order of potency was: neurotransmitter (GABA) > dihydroergotoxine > thiopental > alpha-dihydroergocriptine > dihydroergosine > dihydroergotamine. The results suggest that dihydrogenated ergot derivatives do not bind to the brain benzodiazepine binding sites labeled with 3H-flunitrazepam. However, an enhancement of 3H-flunitrazepam binding by all ergot drugs tested, clearly identifies an allosteric interaction with the benzodiazepine binding sites of GABAA receptors.     

The GABA hypothesis of the pathogenesis of hepatic encephalopathy: current status

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter of the mammalian brain, can induce coma. Outside the central nervous system it is synthesized by gut bacteria and catabolized largely in the liver. GABA and its agonists, as well as benzodiazepines and barbiturates, induce neural inhibition as a consequence of their interaction with specific binding sites for each of these classes of neuroactive substances on the GABA receptor complex of postsynaptic neurons. In a rabbit model of acute liver failure: (i) the pattern of postsynaptic neuronal activity in hepatic coma, as assessed by visual evoked potentials, is identical to that associated with coma induced by drugs which activate the GABA neurotransmitter system (benzodiazepines, barbiturates, and GABA agonists); (ii) the levels of GABA-like activity in peripheral blood plasma increase appreciably before the onset of hepatic encephalopathy, due at least in part to impaired hepatic extraction of gut-derived GABA from portal venous blood; (iii) the blood-brain barrier becomes abnormally permeable to an isomer of GABA, alpha-amino-isobutyric acid, before the onset of hepatic encephalopathy; and (iv) hepatic coma is associated with an increase in the density of receptors for GABA and benzodiazepines in the brain. These findings are the bases of the following hypotheses: (i) when the liver fails, gut-derived GABA in plasma crosses an abnormally permeable blood-brain barrier and by mediating neural inhibition contributes to hepatic encephalopathy; (ii) an increased number of GABA receptors in the brain found in liver failure increases the sensitivity of the brain to GABA-ergic neural inhibition; and (iii) an increased number of drug binding sites mediates the increased sensitivity to benzodiazepines and barbiturates observed in liver failure by permitting increased drug effect.     

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

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.     

Convulsant/depressant site of action at the allosteric benzodiazepine-GABA receptor-ionophore complex

Several classes of centrally acting convulsant, depressant, anticonvulsant and anxiolytic drugs modulate GABAergic transmission. The postsynaptic receptor with which these drugs interact is an allosteric complex with distinct binding sites for GABA, benzodiazepines, picrotoxinin and related compounds. Convulsants which inhibit GABA transmission (except bicuculline) inhibit competitively the binding of dihydropicrotoxinin (DHP) or t-butylbicyclophosphorothionate (TBPT) to the picrotoxinin site and prevent the allosteric enhancing effect of depressant drugs on GABA and benzodiazepine binding. Depressant drugs give a mixed inhibition of TBPT binding. The possible topography of the picrotoxinin site and its relationship to convulsant/depressant drug action at the benzodiazepine-GABA receptor-ionophore complex is discussed.     

Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system.

Barbiturate drugs of diverse chemical structure inhibited the binding of [³H] α-dihydropicrotoxinin to rat brain membranes. This biologically active analoque of picrotoxin labels membrane sites related to the convulsant action of these drugs in inhibiting GABA postsynaptic receptor-ionophore function at a site distinct from the GABA receptor. Depressant barbiturates such as pentobarbital inhibited dihydropicrotoxinin binding competitively at therapeutic concentrations (IC₅₀ = 50 μM) whereas the drug does not alter GABA receptors, uptake, or release at this concentration. Antiepileptics such as phenobarbital (IC₅₀=400 μM), were weaker inhibitors of binding. Convulsant barbiturates, however, such as dimethylbutylbarbiturate (IC₅₀=0.05 μM) and cyclohexylidene-ethyl barbiturate (IC₅₀=0.7 μM), were potent inhibitors. The displacement of radioactive dihydropicrotoxinin binding by the convulsant barbiturates had different slopes and Hill numnbers (0.4) compared to displacement by depressant barbiturates and picrotoxinin itself (Hill numbers = 1.0), indicating heterogeneity of binding sites or negative cooperativity. These potent intractions of barbiturates with dihydropicrotoxinin binding sites are consistent with neurophysiological evidence that depressant or convulsant action of barbiturates may involve modulation of CNS inhibitory synaptic transmission at the level of the postsynaptic GABA receptor-ionophores.     

Anxiety over GABA(A) receptor structure relieved by AChBP

The GABA(A) receptor is the primary mediator of inhibitory neurotransmission in the brain and is a major target for neuromodulatory drugs such as benzodiazepines, barbiturates, ethanol and anaesthetics. However, our understanding of the molecular details of this receptor has been limited by a lack of high-resolution structural information. This article presents a new model for the extracellular, ligand-binding domain of the GABA(A) receptor, that is based on the recently determined structure of a soluble acetylcholine-binding protein. The model puts existing mutational and biochemical data into a three-dimensional context, shows details of the GABA- and benzodiazepine-binding sites, and highlights the importance of other regions in allosteric conformational change. This provides a new perspective on existing data and an exciting new framework for understanding this important family of receptors.     

Pharmacology of GABA-mediated inhibition of spinal cord neurons in vivo and in primary dissociated cell culture

Summary In this paper it is shown that the postsynaptic GABA-receptor chloride ion channel complex is composed of several functional subunits. There are probably at least two stereospecific locations on the receptor for GABA-binding and both must be occupied to obtain an increase in chloride conductance. The interaction between these sites is uncertain but there could be either positive cooperativity between the sites or only a requirement that both sites are occupied without occupation of either site affecting the affinity for GABA of the other site. There is a chloride conductance channel coupled to the GABA receptor which opens for an average of 20 msec and has an average conductance of 18 pS. The GABA-coupled chloride channel may or may not have the same composition as the glycine coupled chloride channel.In addition to the GABA-recognition site and the chloride ion channel, GABA-receptors must have additional binding sites or modulator sites where drugs can bind to modify GABA activation of the GABA-receptor. The convulsant PICRO binds to a site which is independent of the GABA-recognition site and PICRO reduces GABA responses. Barbiturates and benzodiazepines augment GABA-responses without reducing GABA-binding and thus they must bind to a modulator site independent of the GABA recognition site. Whether or not this is the same site as the PICRO binding site is uncertain. Thus, the GABA-receptorchloride ion channel complex is composed of at least: 1) two GABA-binding sites; 2) a chloride ion channel; 3) a convulsant binding site (PICRO-binding site) and 4) an anticonvulsant binding site. This organization serves several obvious purposes. First, since two GABA-molecules are required to activate GABA-coupled chloride ion channels, the dose-response relationship for GABA is sigmoidal and steep. Thus minor shifts in GABA affinity will produce large alterations in GABA-responses and the GABA receptor can be easily modulated. Second, since the receptor has binding sites for convulsant and anticonvulsant compounds which decrease and increase GABA-responses, GABAergic inhibition can easily be modulated.     

Solubilization of convulsant/barbiturate binding activity on the gamma-aminobutyric acid/benzodiazepine receptor complex

Binding activity of the radioactive cage convulsant [35S]t-butylbicyclophosphorothionate was solubilized from rat brain membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio] propanesulfonate. Binding (KD = 26 nM, Bmax = 0.4 pmol/mg protein) was inhibited by picrotoxin and related convulsants and by barbiturates and related depressants that interact with gamma-aminobutyric acid and benzodiazepine receptors via the picrotoxinin binding site. The convulsant/barbiturate binding activity chromatographed on gel filtration as a single peak coinciding with the benzodiazepine/gamma-aminobutyric acid receptor protein complex.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.     

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex

Abstract: Barbiturates enhance the binding of [³H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, K _d , for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [³H]Flunitrazepam binding activity is stable with storage at 4°C., but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly ( t _1/2= 48 h). [³H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.     

Behavioral effects of GABA agonists in relation to anxiety and benzodiazepine action

A considerable body of biochemical and neurophysiological evidence implicates GABA in anxiety and in benzodiazepine action. The present article surveys the behavioral effects of GABA agonists and their interactions with drugs acting at the benzodiazepine receptor in animal anxiety paradigms. Certain GABA agonists, notably valproate, simulate many behavioral actions of benzodiazepines. Moreover, several behavioral studies of the interaction of GABA agonists with benzodiazepines support the hypothesis of a benzodiazepine receptor complex with one or more GABA, benzodiazepine and probably other binding sites. However, there are also a number of anomalous findings of GABA agonist action alone and in combination with benzodiazepines. It is argued that these paradoxical results can better be accounted for in terms of the receptor complex and the distribution of the drugs, rather than by suggesting that the anxiolytic actions of benzodiazepines are not mediated by GABA systems. The potential clinical usefulness of GABA agonists in anxiety is commented upon.     

Relationship of GABAa receptor heterogeneity to regional differences in drug response

Molecular biological approaches to the GABAa receptor have resulted in new insights into the structure and pharmacology of this complex. It is known that the GABAa complex is a heterooligomer composed of multiple subunits which contain binding sites for the GABA, benzodiazepines and barbiturates. These subunits also contain regulatory sites for phosphorylation by intracellular kinases. There appear to be regional differences in the expression of the various subunits for the GABAa receptor complex. The functional significance of molecular heterogeneity is not yet known but it is expected that regional differences may result in pharmacologically diverse responses. Studies on the effects of chronic administration of diazepam have clearly delineated such regional differences. Chronic benzodiazepine administration results in the development of subsensitivity to the electrophysiological actions of GABA in the dorsal raphe, but not in GABA receptive neurons of the substantia nigra pars reticulata. Such data is consistent with regional heterogeneity in response to chronic benzodiazepine, exposure. It is hoped that by understanding GABAa receptor heterogeneity, and its molecular basis, we can improve the, existing receptor subtype specificity and pharmacology of the benzodiazepines.     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

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.     

Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone

The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D_f) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D_f motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.     

Unexpected Properties of δ-Containing GABAA Receptors in Response to Ligands Interacting with the α+ β− Site

GABA_A receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ β? interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αβ, αβγ and αβδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and β subunits present within the receptors. Whereas the presence of a γ2 subunit within αβγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αβ receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1β2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ β? site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ β? interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1β3δ receptors was not observed at α4,6β3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1β3δ receptors was not observed with another α+ β? modulator, and that the efficacy of modulation by α+ β? ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand.