GABAA Receptor-chloride Channel Complex Research Articles

Theoretical study of fluorinated bioisosteres of organochlorine compounds as effective and eco-friendly pesticides.

Chlordane is a worldwide banned organochlorine insecticide because of its hazard to animal and human health. It is also a persistent organic pollutant, which can affect either the soil or the aquatic life. The same applies to other chlorinated cyclodiene insecticides, such as dieldrin and aldrin. In turn, organofluorine compounds have a widespread use in agriculture. Therefore, density functional calculations and docking studies showed that the bioisosteric replacement of chlorines in the above-mentioned compounds by fluorines improves some physicochemical parameters used to estimate the toxicity and environmental risk of these compounds, as well as the ligand-enzyme (GABAA receptor-chloride channel complex) interactions related to their insecticidal activity. This work is an effort to provide an improved new class of organofluorine pesticides.

Exploring the Possible Mode of Anxiolytic Action of a Polyherbal Formulation in Animal Models of Anxiety

The polyherbal formulation, PH-1, containing fresh aqueous extracts of Valeriana and, Boswellia spp. as major constituents and, Plumbago, Acorus spp. as minor constituents was investigated to explore the anti-anxiolytic activity and, its mode of action through competitive GABA receptor bindings with different antagonists of known nature e.g., flumazenil, picrotoxin and bicuculine. The open field, staircase test and, mirror chamber tests of animal models of anxiety were employed at 100, 200, 400 and 800 mg/kg dose levels. The formulation exhibited significant dose dependent anxiolytic activity through oral administration for varying periods of time in different paradigms of increased time-spent in mirror chamber, in central platform of open filed and, the enhanced climbing of steps in staircase tests. The 200 and, 400 mg/kg doses were highly effective. The formulation seems to be acting through GABAA-benzodiazepine binding site as evident from the blockage of PH-1 action at 400 mg/kg dose level by the flumazenil, a known GABAA-benzodiazepine receptor antagonist, and, picrotoxin, a known GABAA receptor-chloride channel complex antagonist. However, the blockade of anxiolytic activity by bicuculine through GABAA-GABA binding receptor antagonist was not significant which also confirmed the mode of action through activation of benzodiazepine receptor and, facilitation of benzodiazepine interdependent chloride channel complex.

Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABA(A) receptors.

The interneurons of the olfactory bulb arise from precursor cells in the anterior part of the neonatal subventricular zone, the SVZa, and are distinctive in that they possess a neuronal phenotype and yet undergo cell division. To characterize the differentiation of neonatal SVZa progenitor cells, we analyzed the complement of ionotropic neurotransmitter receptors that they express in vitro. For this analysis, we tested the sensitivity of SVZa progenitor cells to gamma-amino-n-butyric acid (GABA), adenosine triphosphate (ATP), kainate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) after 1 day in vitro. SVZa progenitor cells had chloride currents activated by GABA and muscimol, the GABA(A) receptor-specific agonist, but were insensitive to ATP, kainate, NMDA, and ACh. In addition, GABA- or muscimol-activated chloride currents were blocked nearly completely by 30 microM bicuculline, the GABA(A) receptor-specific antagonist, suggesting that GABA(B) and GABA(C) receptors are absent. Measurements of the chloride reversal potential by gramicidin-perforated patch clamp revealed that currents generated by activation of GABA(A) receptors were inward, and thus, depolarizing. A set of complementary experiments was undertaken to determine by reverse transcription and polymerase chain reaction (RT-PCR) whether SVZa progenitor cells express the messenger RNA (mRNA) coding for glutamic acid decarboxylase 67 (GAD67), used in the synthesis of GABA and for GABA(A) receptor subunits. Both postnatal day (P0) SVZa and olfactory bulb possessed detectable mRNA coding for GAD67. In P0 SVZa, the GABA(A) receptor subunits detected with RT-PCR included alpha 2-4, beta 1-3, and gamma 2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: alpha 1, alpha 5, gamma 1, gamma 2L (long form), gamma 3, and delta subunit mRNAs. Antibodies recognizing GABA, GAD, and various GABA(A) receptor subunits were used to label SVZa cells harvested from P0-1 rats and cultured for 1 day. The cells were immunoreactive for GABA, GAD, and the GABA(A) receptor subunits alpha 2-5, beta 1-3, and gamma 2. To relate the characteristics of GABA(A) receptors in cultured SVZa precursor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by RT-PCR (alpha 2-4, beta 3, and gamma 2S) were cloned into a mammalian cell expression vector and different combinations were transfected into Chinese hamster ovary-K1 (CHO-K1) cells. A comparison of the sensitivity to inhibition by zinc of GABA(A) receptors in SVZa precursor cells and in CHO-K1 cells expressing various combinations of recombinant GABA(A) receptor subunits suggested that the gamma 2S subunit was present and functional in the GABA(A) receptor chloride channel complex. Thus, SVZa precursor cells are GABAergic and a subset of the GABA(A) receptor subunits detected in the olfactory bulb was found in the SVZa, as might be expected because SVZa progenitor cells migrate to the bulb as they differentiate.

Counteraction of the rapid tolerance to 8-hydroxy-2-(di-n-propylamino)tetralin-induced hypothermia in rats by activation of the GABAA receptor-chloride channel complex.

The effects of compounds that open the GABAA receptor-chloride channel complex on the rapidly developed tolerance to the 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide(8-OH-DPAT)-induced hypothermia in rats were examined. The test compound was injected 15 min. before 1 mg/kg subcutaneous 8-OH-DPAT or saline and 24 hr later a challenge dose of 0.1 mg/kg subcutaneous 8-OH-DPAT was given. The rectal temperature was measured before the challenge dose and 30, 60, 90 and 120 min. thereafter. The hypothermic effect was calculated as the area under the curve. It was found that all the GABAergic compounds examined significantly counteracted the 8-OH-DPAT-induced tolerance to the hypothermic response: muscimol at 3 mg/kg subcutaneous, diazepam at 1 - 3 mg/kg subcutaneous, pentobarbitone sodium at 20 mg/kg subcutaneous, and chlormethiazole at 40 mg/kg subcutaneous. Combined treatment of the rats with the GABAA receptor antagonist, bicucculine, or the GABAA receptor-chloride channel blocker, picrotoxin and diazepam, pentoparbitone sodium or chlormethiazole significantly antagonised this counteraction of the 8-OH-DPAT-induced tolerance. Depletion of 5-HT by pretreatment of the rats with the tryptophan hydroxylase inhibitor p-chlorophenylalanine did not counteract the 8-OH-DPAT-induced tolerance to the hypothermic response. Pretreatment of the rats with dexamethazone did not change the development of the tolerance to 8-OH-DPAT-induced hypothermic effect which seems to exclude the involvement of the hypothalamo-pituitary-adrenocortical axis in the tolerance development. It is concluded that the results support the hypothesis that GABA neurones beyond the 5-HT neurones are involved in the mechanism causing tolerance to the 5-HT1A receptor-mediated hypothermia in rats.

Pharmacological approaches predicting antidepressant or anxiolytic activities: What is common and what is different

Benzodiazepines modulate γ-aminobutyric acid (GABA)-evoked chloride currents through a specific binding site at the GABAA receptor-chloride channel complex. The heterogeneity of diazepam-sensitive benzodiazepine binding sites (type I and type II) has been identified by pharmacological approaches both with native receptors and recombinant receptors coexpressing α, β and γ subunits. In addition, two distinguishable diazepam-insensitive benzodiazepine sites arefound, spatially distributed between cerebral cortical and cerebellar regions. Coexpression of α6 with β2 and γ2L subunits creates a pharmacologically similar benzodiazepine receptor to the diazepam-insensitive site observed in cerebellum, however, there is no evidence regarding the possible subunit combination forming the DI site in cerebral tissues. Here we report the cloning of the human α4 cDNA and its pharmacology by coexpression of this α4 subunit with β2 and γ2L subunits. This recombinant receptor complex showed a high affinity for the previously described benzodiazepine partial agonist bretazenil, the pyrazoloquinoline compounds CGS-9895 and CGS-9896, as well as the inverse agonists DMCM (methyl 6,7-dimethoxy 4-ethyl-β-carboline-3-carboxylate) and Ro15-4513 as determined by [3H]Ro15-4513 binding. However, it is insensitive to the benzodiazepine type I selective compounds CL218,872 (3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine) and zolpidem as well as the benzodiazepine full agonists diazepam, halazolam and midazolam. In addition, the benzodiazepine receptor ligands DMCM, β-CCE (β-carboline-3-carboxylate ethyl ester), β-CCM (β-carboline-3-carboxylate methyl ester), FG-7142, CGS-9895 and CGS-9896 showed 7 to 10 times higher affinity for α4β2γ2L than for α6β2γ2L. The pharmacology of the α4β2γ2L receptor complex appears to resemble those of the diazepam-insensitive site found in the cerebral cortex. Our study thus suggests that this subpopulation of diazepam-insensitive GABAA receptors may be composed of α4β2γ2L subunits.

Neuronal ion channels as the target sites of insecticides.

Certain types of neuronal ions channels have been demonstrated to be the major target sites of insecticides. The insecticide-channel interactions that have been studied most extensively are pyrethroid actions on the voltage-gated sodium channel and cyclodiene/lindane actions on the GABAA receptor chloride channel complex. With the exception of organophosphate and carbamate insecticides which inhibit acetylcholinesterases, most insecticide commercially developed act on the sodium channel and the GABA system. Pyrethroids show the kinetics of both activation and inactivation gates of sodium channels resulting in prolonged openings of individual channels. This causes membrane depolarization, repetitive discharges and synaptic disturbances leading to hyperexcitatory symptoms of poisoning in animals. Only a very small fraction (approximately 1%) of sodium channel population is required to be modified by pyrethroids to produce severe hyperexcitatory symptoms. This toxicity amplification theory applies to pharmacological and toxicological action of other drugs that go through a threshold phenomenon. Selective toxicity of pyrethroids between invertebrates and mammals can be explained based largely on the responses of sodium channels and partly on metabolic degradation. The pyrethroid-sodium channel interaction is also supported by Na+ uptake and batrachotoxin binding experiments. Cyclodienes and lindane exert a dual action on the GABAA system, the initial transient stimulation being followed by a suppression. The stimulation requires the presence of the gamma 2 subunit. The suppression of the GABA system is also documented by Cl- flux and ligand binding experiments. It appears that the sodium channel and the GABA system merit continuing efforts for development of newer and better insecticides. Nitromethylene heterocycles including imidacloprid act on nicotinic acetylcholine receptors. Insect receptors are more sensitive to these compounds than mammalian receptors. Single-channel analyses of the nicotinic acetylcholine receptor of PC12 cells have shown that imidacloprid increases the activity of subconductance state currents and decreases that of main conductance state currents. This may explain the imidacloprid suppression of acetylcholine responses.

Time-Dependent Rundown of GABA Response in Mammalian CNS Neuron During Experimental Anoxia

Gamma-Aminobirtyric acid (GABA) is one of the major neurotransmitters in the mammalian central nervous system (CNS). The activation of post-synaptic GABAA receptor-chloride channel complex is thought to underlie inhibitory postsynaptic potentials ubiquitously in various CNS regions. GABAA receptors are modulated by convulsant, hypnotic-anticonvulsant, anxiolytic and anxiogenic agents and endogenous agents such as nurosteroids and intracellular calcium, ATP, and cyclic AMP. The function of GABAA receptor in CNS neuron is also affected by some pathophysiological processes, e.g., anoxia. For example, it is currently believed that delayed neuronal death after brain ischemia results from excessive cell excitability and/or loss of inhibition. In the present study, we investigated how the GABA-gated chloride current is affected by anoxic conditions. All experiments were carried out on neurons freshly dissociated from rat CNS by the use of both conventional and nystatin perforated patch recording configurations. The GABA response showed a considerable rundown with time in anoxic condition. The rundown was prevented by adding either ouabain or SPAI-I (Na+-K+ ATPase inhibitor-I), suggesting that the experimental anoxia reduced GABA response by decreasing intracellular ATP synthesis. This result was also confirmed by finding that the direct decrease of intracellular ATP concentration using a conventional whole-cell patch recording mode inhibited the GABA-gated chloride response in mammalian CNS neurons.

Multiple actions of dieldrin and lindane on the GABAAreceptor-chloride channel complex of rat dorsal root ganglion neurons

The GABA A receptor-chloride channel complex is known to be the target of cyclodiene insecticides and of γ-hexachlorocyclohexane (γ-HCH, lindane). Dieldrin first enhances, then suppresses, γ-aminobutyric acid (GABA)- induced chloride currents, and the latter action results from the acceleration of desensitization. We now report more detailed analyses of the action of dieldrin and γ-HCH on the GABA A receptor-chloride channel complex of rat dorsal root ganglion, using whole-cell and single-channel patch clamp techniques. Preapplication of dieldrin suppressed GABA-induced chloride currents in a time and dose-dependent manner. No enhancement of current was observed under these conditions, because the enhancing action of dieldrin, which is transient in nature, had ceased by the time GABA-induced currents were recorded. Both transient and sustained components of chloride currents were suppressed by dieIdrin. The effect was almost irreversible, and the current did not recover after 30 min of washout with dieldrin-free solution. The currents activated by higher concentrations of GABA were more strongly suppressed by dieldrin. Coapplication of γ-HCH with GABA caused an enhancement of the GABA-induced chloride currents, which was followed by suppression. Single-channel currents induced by 1 μm GABA were completely suppressed by 1 μm γ-HCH. Hyperexcitation of the nervous system and whole animal caused by dieldrin and γ-HCH is the result of suppression of the GABA receptor-chloride channel complex

GABA receptor-channel complex as a target site of mercury, copper, zinc, and lanthanides.

1. The GABAA receptor-chloride channel complex has been shown to be modulated by a variety of chemicals. Scores of chemicals with diverse and unrelated structures augment the GABA-induced chloride current, while some other chemicals suppress the current. Certain heavy metals and a variety of polyvalent cations increase or decrease the current in a potent and efficacious manner. 2. We have studied the mechanisms whereby mercury, copper, zinc, and lanthanides modulated the GABA system by whole-cell and single-channel patch clamp techniques as applied to the rat dorsal root ganglion neurons in primary culture. 3. Mercuric chloride augmented the GABA-induced current to 115% of control at 0.1 microM and to 270% of control at 100 microM. It also generated a slowly developing inward current carried by a variety of ions. In contrast, methylmercury suppressed the GABA-induced current. The potent stimulation of the GABA system by mercuric chloride is deemed important in mercury intoxication. 4. Copper and zinc suppressed the GABA-induced current with an EC50 of 16 and 19 microM, respectively. They bound to a common site on the external surface of the GABA receptor-channel complex. 5. Lanthanum augmented the GABA-induced current with an EC50 of 230 microM by increasing the affinity of the receptor for GABA. It bound to a site on or near the external surface of the GABA receptor-channel complex which is different from the sites for GABA, barbiturates, benzodiazepines, picrotoxin, and copper/zinc. 6. Six other lanthanides with larger atomic numbers also exerted the same stimulatory effect with their efficacies increasing with the atomic number. 7. Single-channel analyses have revealed that the augmentation of whole-cell current by terbium, a lanthanide, is due to three actions: an increase in the overall mean open time, a decrease in the overall mean closed time, and an increase in the overall mean burst time.

Sex differences in sensitivity to pentylenetetrazol but not in GABAA receptor binding

Female rats have a higher threshold than males for seizures induced by the convulsant pentylenetetrazol, a GABAA receptor-chloride channel complex blocker. No sex difference was observed for the anticonvulsant activities of ethanol or diazepam to protect against pentylenetetrazol seizures. Ovariectomy reduces the pentylenetetrazol seizure threshold of females to that of males. In contrast, females have a lower threshold than males to electroshock seizures. Pentylenetetrazol receptors were compared in males and females and gonadectomized animals by binding of several radioligands to the GABAA receptor complex. No differences were found for these four groups of animals in the binding of [3H]flunitrazepam to the benzodiazepine sites and [35S]t-butyl bicyclophosphorothionate (35S]TBPS) to the cloride channel/convulsant sites in membrane homogenates, nor for allosteric modulation of binding by GABA, the steroid anesthetic alphaxalone, or the benzodiazepine Ro 5-4864. In tissue section autoradiography, no difference was observed for these same assays nor for the binding of [3H]muscimol in the presence and absence of alphaxalone in several major regions. We conclude that circulating female sex hormones, possibly neurosteroid metabolites of progesterone, known to interact directly with the GABAA receptor complex, are involved in the sex differences in pentylenetetrazol seizure susceptibility.

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.

Alcohol, the chloride ionophore and endogenous ligands for benzodiazepine receptors

Considerable evidence suggests that at least some of the effects of ethanol are mediated by an action on the GABAA receptor chloride channel complex. More speculative is the suggestion that ethanol might interact with endogenous ligands for the benzodiazepine receptor on the complex. This paper considers the evidence for such interactions.

Effect of ethanol dependence on GABA A antagonist-induced seizures and agonist-stimulated chloride uptake

The functional state of GABAA receptors during physical dependence on ethanol was evaluated in two ways. First, the ability of ethanol dependence to change the convulsant potency of GABAA antagonists microinjected into the inferior colliculus was examined. A second approach evaluated the effects of ethanol dependence on the ability of muscimol or pentobarbital to stimulate chloride uptake in rat brain vesicles. In the studies examining changes in convulsant potency, bilateral microinfusions of GABAA antagonists, bicuculline methiodide and picrotoxinin, as well as the excitatory amino acid agonist, kainic acid (used as a positive control) induced similar dose-related increases in the frequency of wild-running seizures. Ethanol dependence did not significantly change susceptibility to wild-running seizure induction by an of the convulsants, although susceptibility to the more severe, clonic seizures was significantly increased for each convulsant. This suggested that the receptor-blocking effects of GABAA antagonists responsible for inducing wild-running seizures were not selectively increased by ethanol dependence, but that spread of seizure activity responsible for clonic seizures following the initiation of wild running was generally increased. Finally, in studies examining changes in GABAA receptor-mediated chloride uptake, both muscimol and pentobarbital were found to induce concentration-dependent increases in chloride uptake in rat brain vesicles. However, responses to these drugs were not reduced by ethanol dependence suggesting that a generalized adaptive decrease in GABAA receptor function was unlikely. Together these results do not provide support for the hypothesis that the GABAA receptor-chloride channel complex is down-regulated during the development of physical dependence on ethanol.

The GABA-benzodiazepine interaction fifteen years later

The main steps are presented that led to our current understanding of the interaction between benzodiazepine receptor ligands and the GABAA receptor. The benzodiazepine receptor is a modulatory site located on the GABAA receptor-chloride channel complex that has the unique property of being able to mediate positive as well as negative modulation of the chloride channel gating by the GABAA receptor. Some critical issues concerning the structure of the receptor-channel complex remain to be clarified. Research on the benzodiazepine-GABA interaction has led to novel concepts of drug action and receptor function and provides the basis for a whole spectrum of potential drugs with therapeutic utility.

Lindane but not deltamethrin blocks a component of GABA‐activated chloride channels

Effects of deltamethrin, a pyrethroid insecticide, and lindane, the gamma isomer of hexachlorocyclohexane, on the gamma-aminobutyric acid (GABA)-activated ion channels were studied using cultured neurons isolated from the newborn rat dorsal root ganglia. The neuron was voltage clamped using the whole cell patch clamp technique. Two components of inward chloride currents were generated in response to bath application of GABA. One of the components was selectively blocked by 1 x 10(-5)M lindane in a stereospecific manner, but neither component was affected by 1 x 10(-5)M deltamethrin, which drastically prolonged the voltage-activated sodium channel current. Thus, the action of lindane to stimulate the nervous system is partly because of an interaction with the GABA receptor-channel complex. The target site of deltamethrin is not the GABA receptor-channel complex but the sodium channel. The results suggest a multiplicity of the GABAA receptor-chloride channel complex.