GABAA receptors are differentially sensitive to zinc: dependence on subunit composition.

GABAA receptors with different subunit composition, were expressed in kidney cells and studied by whole cell recording. Expressed GABAA receptors were differentially sensitive to inhibition by zinc; receptors which lacked the gamma subunit were inhibited by zinc. Embryonic neurons also exhibited zinc-sensitive GABA responses, in contrast to adult neurones. This developmentally-sensitive aspect of GABAA receptor pharmacology may be partly dependent on expression of the gamma subunit.     

Enantioselectivity at the physiologically active GABAA receptor.

A subfraction of cortical tissue from rat brain, containing membrane vesicles was prepared freshly with added protease inhibitors and antioxidant. The preparation was used to measure stimulation of transmembrane 36C1- flux and inhibition of bicuculline-sensitive [3H] muscimol binding by (+)-(S) and (-)-(R) enantiomers of dihydromuscimol at 30 degrees in physiological salt solution. Displacement of bound [3H]muscimol and stimulation of 36Cl- flux appeared in the 0.1-10 microM concentration range of the enantiomers, channel gating, however, required rather high concentrations. Degrees of enantioselectivity for channel gating, desensitization of and binding to GABAA receptors were estimated by the concentration ratios of dihydromuscimol enantiomers, [(-)-(R)]/[(+)-(S)], at the same level of response or displacement. Different enantioselectives were observed for channel gating (6 +/- 3), receptor binding (3 +/- 2) and desensitization (no selectivity). The low and concentration-dependent enantioselectives found for channel gating and receptor binding can be explained by desensitization and heterogeneity of GABAA receptors.     

Decreased GABAA receptor subunit mRNA concentrations following chronic lorazepam administration.

Chronic benzodiazepine administration has been associated with alterations in binding and function at the GABAA receptor. To evaluate effects of chronic benzodiazepine exposure on messenger RNA (mRNA) concentrations for several GABAA receptor subunits, we treated mice with lorazepam, 2 mg kg-1 daily for 1-28 days and evaluated mRNAs for the alpha 1 and gamma 2 subunits by Northern hybridization. In cerebral cortex, concentrations of mRNA for the alpha 1 and gamma 2 subunits were unchanged from vehicle or control after 1-10 days of lorazepam. However, after 14 days of treatment mRNA concentrations for both subunits decreased to approximately 50% of control values and remained decreased at 28 days. In contrast, no significant alterations were observed for either subunit mRNA in hippocampus or cerebellum over the same time course. Alterations in mRNAs in cortex occur after the development of tolerance and receptor downregulation in this model.     

Temperature and anion dependence of allosteric interactions at the gamma-aminobutyric acid-benzodiazepine receptor.

The temperature dependence of [3H]flunitrazepam ([3H]FNZ) binding to rat brain membranes was examined in the presence of the anaesthetics, pentobarbitone, alphaxalone and propofol. Van't Hoff plots showed the binding of FNZ to be largely enthalpy driven. Alphaxalone and propofol increased the entropy of the binding reaction but not the enthalpy and therefore did not show temperature dependence in their efficacy. In contrast, pentobarbitone increased the enthalpy of FNZ binding and, therefore, is more efficacious at low temperatures. The EC50 values of all three modulators increased with temperature indicating that their interactions with the receptor may be enthalpy driven. The EC50 values of all three modulators were also anion dependent, showing a decrease in the presence of gamma-aminobutyric acid (GABAA)-channel permeant anions. The efficacies of alphaxalone and pentobarbitone, but not that of propofol, also increased with increasing chloride ion concentration. The results indicate that all three modulators interact with the GABAA receptor at distinct recognition sites.     

Pharmacological modulation of adrenal medullary GABAA receptor: consistent with its subunit composition.

1. Muscimol, the specific GABAA receptor agonist, increased the secretion of catecholamines by chromaffin cells with an EC50 of 2.9 +/- 0.4 microM. 2. GABAA receptors of these cells were modulated by the same drugs which modulate GABAA receptors in brain tissue. 3. Benzodiazepines enhanced muscimol-evoked catecholamine secretion by between 20 and 80%. This effect seems to be mediated by binding to a central type of benzodiazepine receptor because it was completely blocked by the specific antagonist, Ro 15 1788. This antagonist was able to displace [3H]-flunitrazepam binding with an EC50 of 0.26 +/- 0.05 nM. 4. beta-Carbolines weakly inhibited muscimol-induced catecholamine secretion and were able to displace [3H]-flunitrazepam binding with an EC50 between 0.2 and 0.9 nM, depending on the beta-carboline used. 5. Pregnanolone and related neuroactive steroids enhanced muscimol-evoked catecholamine secretion by up to 87%, in a dose-dependent fashion. In contrast pregnenolone weakly inhibited muscimol-evoked catecholamine secretion. 6. Zn2+ did not affect GABAA receptor-induced catecholamine secretion. 7. These pharmacological results are absolutely concordant with the theoretical properties given by the GABAA receptor subunit composition of bovine adrenal medulla -alpha 1, alpha 4, beta 1-3, gamma 2-previously characterized by Western blot analysis.     

Effects of gamma-HCH and delta-HCH on human recombinant GABA(A) receptors: dependence on GABA(A) receptor subunit combination

1. Human GABA(A) receptors containing different alpha and beta subunits with or without the gamma 2S or gamma 2L subunits were expressed in XENOPUS: oocytes and the effects of the insecticides gamma- and delta-hexachlorocyclohexane (gamma-HCH and delta-HCH, respectively) on these receptor subunit combinations were examined using two electrode voltage-clamp procedures. 2. gamma-HCH produced incomplete inhibition of GABA responses on all receptor combinations examined with affinities in the range of 1.1--1.9 microM. Affinity was not dependent on subunit composition but the maximum percentage of inhibition was significantly reduced in beta 1-containing receptors. delta-HCH both potentiated GABA(A) receptors and activated them in the absence of GABA at concentrations higher than those producing potentiation. Allosteric enhancement of GABA(A) receptor function by delta-HCH was not affected by the subunit composition of the receptor, By contrast the GABA mimetic actions of delta-HCH were abolished in receptors containing either alpha 4, beta 1 or gamma 2L subunits. 4. Sensitivity to the direct actions were not restored in receptors containing the mutant beta 1(S290N) subunit, but alpha 1 beta 2 gamma 2L receptors became sensitive to the direct actions of delta-HCH when oocytes were treated for 24 h with the protein kinase inhibitor isoquinolinesulphonyl-2-methyl piperazine dihydrochloride (H-7). 5. We have shown the influence of various alpha, beta and gamma subunits on the inhibitory, GABA mimetic and allosteric effects of HCH isomers. The data reveal that neither the inhibitory actions of gamma-HCH nor the allosteric effects delta-HCH has a strict subunit dependency. By contrast, sensitivity to the direct actions of delta-HCH are abolished in receptors containing alpha 4, beta 1 or gamma 2L subunits.     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Effects of subunit types of the cloned GABAA receptor on the response to a neurosteroid.

Combinations of cloned GABAA receptor subtypes, having the subunit combinations alpha i + beta 1 or alpha i + beta 1 + gamma 2 (i = 1, 2, 3), were expressed in Xenopus oocytes. The endogenous steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiates GABA currents induced therein by GABA. This potentiation was greater in the alpha 1 + beta and alpha 3 + beta 1 than in the alpha 2 + beta 1 combinations. The presence of the gamma 2-subunit increased the steroid potency in alpha 1 + beta 1 and alpha 2 + beta 1, but the combination alpha 3 + beta 1 + gamma 2 became much less steroid-sensitive. It is concluded that the steroid modification of the GABAA receptor is strongly influenced by the alpha- and the gamma 2-subunit types.     

Effects of gamma2S subunit incorporation on GABAA receptor macroscopic kinetics

GABA(A) receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the gamma subunit to the kinetic properties of GABA(A) receptors. Studies in Xenopus oocytes have suggested that co-expression of alpha1, beta2, and gamma 2S subunits results in the formation of both alpha beta and alpha betagamma receptors (Boileau et al. 2002a; Boileau et al., 1998). Here, we have used an excess of the gamma 2S subunit in transfections of HEK293 cells to bias expression toward alpha beta gamma-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the gamma subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, alpha betagamma receptors had an increased GABA EC(50), reduced sensitivity to block by Zn(2+), and did not display outward rectification as compared to alpha beta receptors.     

Mechanism of psychoactive drug action in the brain: simulation modeling of GABAA receptor interactions at non-equilibrium conditions

Synaptic transmission requires that the binding of the transmitter to the receptor to occur under rapidly changing transmitter levels, and this binding interaction is unlikely to be at equilibrium. We have sought to numerically solve for binding kinetics using ordinary differential equations and simultaneous difference equations for use in stochastic conditions. The reaction scheme of GABA interacting with the ligand-gated ion-channel demonstrates numerical stiffness. Implicit methods (Backward Euler, ode23s) performed orders of magnitude better than explicit methods (Forward Euler, ode23, RK4, ode45) in terms of step size required for stability, number of steps and cpu time. Interestingly, upon solving the system of 8 ordinary differential equations for the GABA reaction scheme we observed the existence of low dimensional invariant manifolds that may have important consequences for information processing in synapses. We also describe a mathematical approach that models complex receptor interactions in which the timing and amplitude of transmitter release are noisy. Exact solutions for simple bimolecular interactions that include stoichiometric interactions and receptor transitions can be used to model complex reaction schemes. We used the difference method to investigate the information processing capabilities of GABA(A) receptors and to predict how pharmacological agents may modify these properties. Initial simulations using a model for heterosynaptic regulation shows that signal to noise ratios can be decreased in the presence of background presynaptic activity both in the presence and absence of chlorpromazine. These types of simulations provide a platform for investigating the effect of psycho-active drugs on complex responses of transmitter-receptor interactions in noisy cellular environments such as the synapse. Understanding this process of transmitter-receptor interactions may be useful in the development of more specific and highly targeted modes of action.     

Aging reduces the mRNA of alpha 1 GABAA 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 alpha subunit mRNA level of GABAA 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, alpha 1 mRNA level was markedly decreased (86% suppression). In contrast, alpha 1 mRNA remained unchanged in cerebellum. These findings indicate a selective age-related structural change in GABAA receptor in rat cerebral cortex.     

A mutation in the GABAA receptor alpha 1 subunit linked to human epilepsy affects channel gating properties

A genetic component is associated with the development of many forms of epilepsy. Recently, mutations in the GABAA receptor have been linked to several inherited epilepsies. One of these mutations is a non-conservative change of alanine to aspartate in the third transmembrane domain of the alpha1 subunit. To determine the functional consequences of this alteration, mutated alpha subunits were transiently transfected along with wild-type beta3 and gamma2L subunits into HEK-293T cells. The mutated alpha1(A294D) subunit reduced GABA sensitivity of the receptor, increased the deactivation rate and slowed desensitization. The mutation caused a reduction in channel open time but no change in single channel conductance. Studies with additional mutants, altering the charge and/or size of the side-chain, indicated that both size and hydrophobicity of the residue at this location influence channel gating. The effects on GABA sensitivity, deactivation rate and channel open time are consistent with a reduced efficacy of channel gating, and would be expected to decrease GABAergic neurotransmission. The alpha1 subtype is the most widely expressed of the alpha subunits, with expression increasing throughout development. Therefore, production of the mutated subunit could cause global hyperexcitability throughout the brain, leading to generalized seizures with juvenile onset.     

The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation.

1. Whole-cell patch clamp recordings were made from L-cells transfected with 2 combinations of subunits of the GABAA receptor. Log concentration-response curves were constructed to gamma-aminobutyric acid (GABA) on alpha 1,beta 1,gamma 2L containing cells and compared to those from alpha 1,beta 1 containing cells. The effects of flunitrazepam, pentobarbitone and alphaxalone on the concentration-response relationships were also examined. 2. From the log concentration-response curves, GABA had a mean (+/- s.e. mean) pEC50 = 5.2 +/- 0.09 and slope factor = 1.7 +/- 0.02 on alpha 1,beta 1,gamma 2L cells which were significantly different from the values obtained from alpha 1,beta 1 cells where the pEC50 = 5.6 +/- 0.02 and the slope = 1.5 +/- 0.02. 3. Flunitrazepam produced a parallel leftward shift of GABA concentration-response curves on alpha 1,beta 1,gamma 2L cells. The EC50 for flunitrazepam = 6.3 +/- 2.7 nM. No increase in the maxima of the GABA concentration-response curves was found in the presence of flunitrazepam. Flunitrazepam did not potentiate responses from alpha 1,beta 1 cells. 4. The log concentration-response curves from both populations of cells were shifted to the left by equal amounts by pentobarbitone. A significant increase in the maximal response to GABA was also produced by pentobarbitone. This occurred at lower concentrations of pentobarbitone on alpha 1,beta 1 cells. 5. Alphaxalone produced leftward shifts of GABA log concentration-response curves of similar magnitudes in both populations of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid

When vertebrate brain poly(A)⁺ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs enconding bovine GABA_A receptor subunits in the combinations α1 + ß1, or αi + ß1 + γ2 (where i = 1. 2 or 3). The characteristics of this inhibition vary significantly with the type of the α subunit (α1, α2, or α3) used. When the bovine γ2L alternate form of the γ2 subunit was replaced by the human γ2S subunit, the behaviour was unchanged: the human γ2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human γ2 subunit previously described. The results of co-application of PS and 3α-hydroxy-5α-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABA_A receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is α-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.     

Barbiturate physical dependence in mice: effects of neuroleptics and diazepam on the withdrawal syndrome.

Diazepam (8 mg/kg) quickly and effectively reduced the incidence of withdrawal symptoms in phenobarbital dependent C57BL/6J male mice. In contrast, the neuroleptic agents employed (chlorpormazine, haloperidol, or reserpine) or alpha-methyl-p-tyrosine tended to exacerbate the withdrawal syndrome. Chlorpromazine and haloperidol were approximately equally potent in their effects; this would suggest that their antidopaminergic effects were not the primary mechanism of action for the increased incidence of withdrawal symptoms.     

Juvenile stress-induced alteration of maturation of the GABAA receptor alpha subunit in the rat

Profound evidence indicates that GABAA receptors are important in the control of physiological response to stress and anxiety. The alpha subunit type composition contributes significantly to the functional characterization of the GABAA receptors. The alpha2, alpha3, alpha5 subunits are predominately expressed in the brain during embryonic and early postnatal periods of normal rats, whilst alpha1 are most prominent during later developmental stages. In the present study, we examined the long-term effects of juvenile stress on GABA alpha subunit expression in adulthood in the amygdala and hippocampus. We applied the elevated platform stress paradigm at juvenility and used the open-field and startle response tests to assess anxiety level in adulthood. Juvenile stress effects without behavioural tests in adulthood were also examined since previous studies indicated that the mere exposure to these tests might be stressful for rats, enhancing the effects of the juvenile exposure to stress. In adulthood, we quantitatively determined the level of expression of alpha1, alpha2 and alpha3 in the hippocampus and amygdala. Our results indicate that subjecting juvenile stressed rats to additional challenges in adulthood results in an immature-like expression profile of these subunits. To test for potential functional implications of these alterations we examined the effects of the anxiolytic (diazepam) and the sedative (brotizolam) benzodiazepines on juvenile stressed and control rats following additional challenges in adulthood. Juvenile stressed rats were more sensitive to diazepam and less sensitive to brotizolam, suggesting that the alterations in GABA alpha subunit expression in these animals have functional consequences.     

Barbiturate dependence in mice: Effects of continuous vs. Discontinuous drug administration

Three groups of DBA/2J mice were continuously exposed for 3, 6 or 9 days to a milled diet containing phenobarbital. Two additional groups were given a discontinuous drug administration schedule with either one or two 24 h drug-free periods interpolated among the 6 or 9 days of phenobarbital consumption, respectively. The discontinuous schedule of drug administration significantly attenuated the development of physical dependence as determined by the severity of withdrawal symptoms. Functional tolerance development was also attenuated in a manner that closely paralleled the effects on physical dependence development. The 5 group means showed a perfect rank order correlation between functional tolerance and physical dependence development.     

GABAA receptor subunit mRNA levels are differentially influenced by chronic FG 7142 and diazepam exposure.

Levels of mRNA for the alpha 1, gamma 2 and beta 1 subunits of the GABAA receptor complex were examined in rats maintained on a chronic, continuous schedule of exposure to the benzodiazepine inverse agonist FG 7142. The effect of chronic exposure to the benzodiazepine agonist diazepam was also examined on levels of gamma 2 subunit mRNA. FG 7142 (2 mg/ml of 100% dimethyl sulfoxide (DMSO) or vehicle (100% DMSO) was administered continuously for 8 days in the right ventricle via an osmotic minipump. At the end of the eighth day of exposure, the brain was removed and cerebral cortex, cerebellum and hippocampus were dissected and mRNA prepared from each region. Levels of GABAA alpha 1 and gamma 2 subunit mRNA were examined by Northern blot analysis with cDNA probes specific for these subunits. A significant increase in alpha 1 mRNA was measured in both cortex and hippocampus, but not in cerebellum, of rats chronically exposed to FG 7142 relative to vehicle-treated rats. A significant increase in gamma 2 subunit mRNA in cortex was also evident in drug-treated rats; however, no change in gamma 2 subunit mRNA was observed in either the hippocampus or cerebellum. Examination of GABAA beta 1 subunit mRNA by solution hybridization using a beta 1 riboprobe revealed no effect of chronic FG 7142 treatment on this subunit in either cortex, hippocampus or cerebellum. In rats chronically exposed to diazepam (21 days via silastic implants), levels of gamma 2 subunit mRNA were significantly decreased in cortex, but not changed in either hippocampus or cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of membrane cholesterol on the GABAA receptor.

1. Neurosteroids such as pregnanolone have been established as potent modulators of the GABAA receptor in both electrophysiological and binding studies. Since cholesterol is present in substantial amounts in the neuronal membranes, we have sought evidence for possible interactions of cholesterol with the neurosteroid site and more generally, with the GABAA channel. 2. Synaptosomal membranes were prepared from rat whole brain, cerebral cortex, cerebellum and spinal cord. These membranes were enriched with cholesterol to about double the original level by incubation with liposomes comprised of 50 phosphatidylcholine: 50 cholesterol in the presence of 1% BSA. The additional cholesterol formed a homogeneous mixture with the endogenous cholesterol. 3. The effects of cholesterol and modulatory drugs on the GABAA channel were assessed from the changes induced in [3H]-flunitrazepam (FNZ) binding. Cholesterol enrichment did not affect FNZ binding itself; however, the enhancement of [3H]-FNZ binding by pregnanolone was affected. In membranes from cerebral cortex, the potency of pregnanolone was reduced by a factor of 3.2 following cholesterol enrichment. By contrast, in membranes from spinal cord, the potency of pregnanolone was increased by a factor of 8.4 following cholesterol enrichment. In membranes from cerebellum, there was little overall change in pregnanolone potency although the effects of threshold concentrations were increased. 4. The enhancement of [3H]-FNZ binding by propofol in whole brain membranes was reduced in cholesterol-enriched membranes, similar to the effects of pregnanolone. Experiments with muscimol resulted in an increase in its potency as a potentiator of [3H]-FNZ binding, following cholesterol enrichment. 5. These results provide little evidence for a selective competition between cholesterol and pregnanolone at its binding site. Rather, they suggest an influence of membrane cholesterol on the functional coupling between the benzodiazepine site and the other specific drug sites on the GABAA channel. The detailed pattern of influence depended upon the region of CNS and may be related to the subunit composition of the GABAA channels present.