Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

How does α1Histidine102 affect the binding of modulators to α1ß2γ2 GABAA receptors? molecular insights from in silico experiments.

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Interaction of GABAA and GABAB antagonists after status epilepticus in immature rats.

Among neurotransmitter systems affected by status epilepticus (SE) in adult rats are both GABAergic systems. To analyze possible changes of GABAA and GABAB systems in developing rats lithium-pilocarpine SE was induced at postnatal day 12 (P12). Seizures were elicited by a GABAA antagonist pentylenetetrazol (PTZ) 3, 6, 9, and 13 days after SE (i.e., in P15, P18, P21, and P25 rats), and their possible potentiation by a GABAB receptor antagonist CGP46381 was studied. Pilocarpine was replaced by saline in control animals (lithium-paraldehyde [LiPAR]). Pentylenetetrazol in a dose of 50 mg/kg s.c. elicited generalized seizures in nearly all 15-day-old naive rats and in 40% of 18-day-old ones but not in older animals. After SE, PTZ no longer elicited seizures in these two younger groups, i.e., sensitivity of GABAA system was diminished. The GABAB antagonist exhibited proconvulsant effect in P15 and P18 SE as well as LiPAR rats returning the incidence of PTZ-induced seizures to values of control animals. A decrease in the incidence of minimal clonic seizures was seen in P21 LiPAR animals; these seizures in the oldest group were not affected. Change of the effect from proconvulsant to anticonvulsant (or at least to no action) took place before postnatal day 21. Both SE and LiPAR animals exhibited similar changes but their intensity differed, effects in LiPAR controls were usually more expressed than in SE rats.

Design of a Highly Bistable Photoswitchable Tethered Ligand for Rapid and Sustained Manipulation of Neurotransmission.

Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. However, their applications for slow or long-lasting biological events are constrained by fast thermal relaxation of cis-azobenzene. We address this issue by modifying the ortho positions of azobenzene used in the tethered ligand. In cultured cells and intact brain tissue, conjugating inhibitory neurotransmitter receptors with one of the derivatives, dMPC1, allows bidirectional receptor control with 380 and 500 nm light. Moreover, the receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales.

GABAA receptor target of tetramethylenedisulfotetramine.

Use of the highly toxic and easily prepared rodenticide tetramethylenedisulfotetramine (TETS) was banned after thousands of accidental or intentional human poisonings, but it is of continued concern as a chemical threat agent. TETS is a noncompetitive blocker of the GABA type A receptor (GABAAR), but its molecular interaction has not been directly established for lack of a suitable radioligand to localize the binding site. We synthesized [(14)C]TETS (14 mCi/mmol, radiochemical purity >99%) by reacting sulfamide with H(14)CHO and s-trioxane then completion of the sequential cyclization with excess HCHO. The outstanding radiocarbon sensitivity of accelerator mass spectrometry (AMS) allowed the use of [(14)C]TETS in neuroreceptor binding studies with rat brain membranes in comparison with the standard GABAAR radioligand 4'-ethynyl-4-n-[(3)H]propylbicycloorthobenzoate ([(3)H]EBOB) (46 Ci/mmol), illustrating the use of AMS for characterizing the binding sites of high-affinity (14)C radioligands. Fourteen noncompetitive antagonists of widely diverse chemotypes assayed at 1 or 10 µM inhibited [(14)C]TETS and [(3)H]EBOB binding to a similar extent (r(2) = 0.71). Molecular dynamics simulations of these 14 toxicants in the pore region of the α1ß2γ2 GABAAR predict unique and significant polar interactions for TETS with α1T1' and γ2S2', which are not observed for EBOB or the GABAergic insecticides. Several GABAAR modulators similarly inhibited [(14)C]TETS and [(3)H]EBOB binding, including midazolam, flurazepam, avermectin Ba1, baclofen, isoguvacine, and propofol, at 1 or 10 µM, providing an in vitro system for recognizing candidate antidotes.

Cloaked Caged Compounds: Chemical Probes for Two-Photon Optoneurobiology.

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time.

Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7-Nitroindolinyl- and 2-(p-Phenyl-o-nitrophenyl)propyl-Based Photochemical Probes.

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or ß2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2ß2 as well as more physiologically relevant α2ß2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and ß2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

Efficacy of synaptic inhibition depends on multiple, dynamically interacting mechanisms implicated in chloride homeostasis.

Chloride homeostasis is a critical determinant of the strength and robustness of inhibition mediated by GABA(A) receptors (GABA(A)Rs). The impact of changes in steady state Cl(-) gradient is relatively straightforward to understand, but how dynamic interplay between Cl(-) influx, diffusion, extrusion and interaction with other ion species affects synaptic signaling remains uncertain. Here we used electrodiffusion modeling to investigate the nonlinear interactions between these processes. Results demonstrate that diffusion is crucial for redistributing intracellular Cl(-) load on a fast time scale, whereas Cl(-)extrusion controls steady state levels. Interaction between diffusion and extrusion can result in a somato-dendritic Cl(-) gradient even when KCC2 is distributed uniformly across the cell. Reducing KCC2 activity led to decreased efficacy of GABA(A)R-mediated inhibition, but increasing GABA(A)R input failed to fully compensate for this form of disinhibition because of activity-dependent accumulation of Cl(-). Furthermore, if spiking persisted despite the presence of GABA(A)R input, Cl(-) accumulation became accelerated because of the large Cl(-) driving force that occurs during spikes. The resulting positive feedback loop caused catastrophic failure of inhibition. Simulations also revealed other feedback loops, such as competition between Cl(-) and pH regulation. Several model predictions were tested and confirmed by [Cl(-)](i) imaging experiments. Our study has thus uncovered how Cl(-) regulation depends on a multiplicity of dynamically interacting mechanisms. Furthermore, the model revealed that enhancing KCC2 activity beyond normal levels did not negatively impact firing frequency or cause overt extracellular K(-) accumulation, demonstrating that enhancing KCC2 activity is a valid strategy for therapeutic intervention.

Hypnotic effects and GABAergic mechanism of licorice (Glycyrrhiza glabra) ethanol extract and its major flavonoid constituent glabrol.

Licorice (Glycyrrhiza glabra, GG) is one of the most frequently used herbal medicines worldwide, and its various biological activities have been widely studied. GG is reported to have neurological properties such as antidepressant, anxiolytic, and anticonvulsant effects. However, its hypnotic effects and the mechanism of GG and its active compounds have not yet been demonstrated. In this study, GG ethanol extract (GGE) dose-dependently potentiated pentobarbital-induced sleep and increased the amount of non-rapid eye movement sleep in mice without decreasing delta activity. The hypnotic effect of GGE was completely inhibited by flumazenil, which is a well-known γ-aminobutyric acid type A-benzodiazepine (GABA(A)-BZD) receptor antagonist, similar to other GABA(A)-BZD receptor agonists (e.g., diazepam and zolpidem). The major flavonoid glabrol was isolated from the flavonoid-rich fraction of GGE; it inhibited [(3)H] flumazenil binding to the GABA(A)-BZD receptors in rat cerebral cortex membrane with a binding affinity (K(i)) of 1.63 µM. The molecular structure and pharmacophore model of glabrol and liquiritigenin indicate that the isoprenyl groups of glabrol may play a key role in binding to GABA(A)-BZD receptors. Glabrol increased sleep duration and decreased sleep latency in a dose-dependent manner (5, 10, 25, and 50mg/kg); its hypnotic effect was also blocked by flumazenil. The results imply that GGE and its flavonoid glabrol induce sleep via a positive allosteric modulation of GABA(A)-BZD receptors.

2-Aminoethyl methylphosphonate, a potent and rapidly acting antagonist of GABA(A)-ρ1 receptors.

2-Aminoethyl methylphosphonate (2-AEMP), an analog of GABA, has been found to exhibit antagonist activity at GABA(A)-ρ1 (also known as ρ1 GABA(C)) receptors. The present study was undertaken to elucidate 2-AEMP's action and to test the activities of 2-AEMP analogs. Whole-cell patch-clamp techniques were used to record membrane currents in neuroblastoma cells stably transfected with human GABA(A)-ρ1 receptors. The action of 2-AEMP was compared with that of 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (TPMPA), a commonly used GABA(A)-ρ1 antagonist. With 10 µM GABA, 2-AEMP's IC(50) (18 µM) differed by less than 2.5-fold from that of TPMPA (7 µM), and results obtained were consistent with a primarily competitive mode of inhibition by 2-AEMP. Terminating the presentation of 2-AEMP or TPMPA in the presence of GABA produced a release from inhibition. However, the rate of inhibition release upon the termination of 2-AEMP considerably exceeded that determined with termination of TPMPA. Moreover, when presented at concentrations near their respective IC(50) values, the preincubation period associated with 2-AEMP's onset of inhibition was much shorter than that for TPMPA. Analogs of 2-AEMP possessing a benzyl or n-butyl rather than a methyl substituent at the phosphorus atom, as well as analogs bearing a C-methyl substituent on the aminoethyl side chain, exhibited reduced potency relative to 2-AEMP. Of these analogs, only (R)-2-aminopropyl methylphosphonate significantly diminished the response to 10 µM GABA. Structure-activity relationships are discussed in the context of molecular modeling of ligand binding to the antagonist binding site of the GABA(A)-ρ1 receptor.

Central efferent pathways for cold-defensive and febrile shivering.

Shivering is a remarkable somatomotor thermogenic response that is controlled by brain mechanisms. We recorded EMGs in anaesthetized rats to elucidate the central neural circuitry for shivering and identified several brain regions whose thermoregulatory neurons comprise the efferent pathway driving shivering responses to skin cooling and pyrogenic stimulation. We simultaneously monitored parameters from sympathetic effectors: brown adipose tissue (BAT) temperature for non-shivering thermogenesis and arterial pressure and heart rate for cardiovascular responses. Acute skin cooling consistently increased EMG, BAT temperature and heart rate and these responses were eliminated by inhibition of neurons in the median preoptic nucleus (MnPO) with nanoinjection of muscimol. Stimulation of the MnPO evoked shivering, BAT thermogenesis and tachycardia, which were all reversed by antagonizing GABA(A) receptors in the medial preoptic area (MPO). Inhibition of neurons in the dorsomedial hypothalamus (DMH) or rostral raphe pallidus nucleus (rRPa) with muscimol or activation of 5-HT1A receptors in the rRPa with 8-OH-DPAT eliminated the shivering, BAT thermogenic, tachycardic and pressor responses evoked by skin cooling or by nanoinjection of prostaglandin (PG) E2, a pyrogenic mediator, into the MPO. These data are summarized with a schematic model in which the shivering as well as the sympathetic responses for cold defence and fever are driven by descending excitatory signalling through the DMH and the rRPa, which is under a tonic inhibitory control from a local circuit in the preoptic area. These results provide the interesting notion that, under the demand for increasing levels of heat production, parallel central efferent pathways control the somatic and sympathetic motor systems to drive thermogenesis.

GABAA receptor open-state conformation determines non-competitive antagonist binding.

The γ-aminobutyric acid (GABA) type A receptor (GABA(A)R) is one of the most important targets for insecticide action. The human recombinant ß3 homomer is the best available model for this binding site and 4-n-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the ß3 homomer relative to the much-less-active but structurally very-similar ß1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The ß1 and ß3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera ß3/ß1 with the ß3 subunit extracellular domain and the ß1 subunit transmembrane helices retained the high [(3)H]EBOB binding level of the ß3 homomer while chimera ß1/ß3 with the ß1 subunit extracellular domain and the ß3 subunit transmembrane helices had low binding activity similar to the ß1 homomer. GABA at 3µM stimulated heteromers α1ß1 and α1ß3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive ß1/ß3 chimera close to wildtype α1ß1 activity. EBOB binding was significantly altered by mutations ß1S15'N and ß3N15'S compared with wildtype ß1 and ß3, respectively. However, the binding activity of α1ß1S15'N was insensitive to GABA and α1ß3N15'S was stimulated much less than wildtype α1ß3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation ß3N15'S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA(A) receptor sensitivity.

Synthesis and testing of a p-H2 hyperpolarized 13C probe based on the pyrazolo[1,5-a]pyrimidineacetamide DPA-713, an MRI vector to target the peripheral benzodiazepine receptors.

DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5-a]pyrimidineacetamide series that has been shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator protein (18 kDa) or in short TSPO. On the basis of this structure, a novel derivative bearing a [(13)C]butynoate moiety has been designed and synthesized (three steps-42% overall yield) providing, upon rapid and quantitative para-hydrogenation, the corresponding hyperpolarized [(13)C]alkene. Para-hydrogen-induced polarization effects have been detected in both (1)H and (13)C-NMR spectra. Upon applying a field cycling procedure, the spin order of para-H(2) added hydrogens is transferred on the (13)C carboxylate moiety yielding a signal enhancement of approximately 4500 times. T(1) of the carboxylate carbon atom is approximately 21.9 s (at 9.37 T). A (13)C-MR image has been acquired by using the (13)C RARE (Rapid Acquisition by Relaxation Enhancement) acquisition protocol on a 10-mM solution. The main limitation to the in vivo use of this novel para-hydrogenated [(13)C]derivative is its relatively low solubility in aqueous systems.

GABA(A) receptor antagonism increases NMDA receptor inhibition by isoflurane at a minimum alveolar concentration.

OBJECTIVE: At the minimum alveolar concentration (MAC), isoflurane potentiates GABA(A) receptor currents and inhibits NMDA receptor currents, and these actions may be important for producing anesthesia. However, isoflurane modulates GABA(A) receptors more potently than NMDA receptors. The objective of this study was to test whether isoflurane would function as a more potent NMDA receptor antagonist if its efficacy at GABA(A) receptors was decreased. STUDY DESIGN: Prospective experimental study. ANIMALS: Fourteen 10-week-old male Sprague-Dawley rats weighing 269 ± 12 g. METHODS: Indwelling lumbar subarachnoid catheters were surgically placed in isoflurane-anesthetized rats. Two days later, the rats were anesthetized with isoflurane, and artificial CSF containing either 0 or 1 mg kg(-1) picrotoxin, a GABA(A) receptor antagonist, was infused intrathecally at 1 µL minute(-1). The baseline isoflurane MAC was then determined using a standard tail clamp technique. MK801 (dizocilpine), an NMDA receptor antagonist, was then administered intravenously at 0.5 mg kg(-1). Isoflurane MAC was re-measured. RESULTS: Picrotoxin increased isoflurane MAC by 16% compared to controls. MK801 significantly decreased isoflurane MAC by 0.72% of an atmosphere in controls versus 0.47% of an atmosphere in rats receiving intrathecal picrotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A smaller MK801 MAC-sparing effect in the picrotoxin group is consistent with greater NMDA antagonism by isoflurane in these animals, since it suggests that fewer NMDA receptors are available upon which MK801 could act to decrease isoflurane MAC. Decreasing isoflurane GABA(A) potentiation increases isoflurane NMDA antagonism at MAC. Hence, the magnitude of an anesthetic effect on a given channel or receptor at MAC may depend upon effects at other receptors.

Microwave Assisted Synthesis and Molecular Docking Studies of Some 4- (3H)-quinazolinone Derivatives as Inhibitors of Human Gamma- Aminobutyric Acid Receptor, the GABA (A)R-BETA3 Homopentamer.

BACKGROUND: Quinazolines and quinazolinones constitute a major class of biologically active molecules, both from natural and synthetic sources. The quinazolinone moiety is an important pharmacophore showing many types of pharmacological activities as shown in recent exhaustive review on the chemistry of 2-heteroaryl & heteroalkyl-4-quinazolinones4-quinazolinones that are the formal condensation products of anthranilic acid and amides. They can also be prepared in this fashion through the Niementowski quinazolinone synthesis, named after it's discoverer Stefan Niementowski. Quinazoline and condensed quinazoline exhibit potent central nervous system (CNS) activities like anti-anxiety, analgesic, anti-inflammatory and anticonvulsant. Quinazolin-4- ones with 2, 3-disubstitution is reported to possess significant analgesic, anti-inflammatory and anticonvulsant activities. METHODS: To expand these views and application profiles, efforts have been made for the synthesis of a new class of quinazolinone by incorporating different amines into synthesized benzoxazinone ring by replacing O atom in the ring. Up till now, a great number of various procedures have been proposed for the synthesis of quinazolin-4-ones in the past few years. Using microwave radiation, this reaction could be easily and rapidly performed in very good yields, providing a large number of various 3-substituted-2- propyl-quinazolin-4-one derivatives which can be employed as useful bioactive compounds. We report a facile and efficient method for the synthesis of 3-substituted-2- propyl-quinazolin-4-one by the condensation reaction of anthranilic acid or halogen substituted anthranilic acid or methyl anthranilate, butanoic anhydride with various amines. We also report a drug/ligand or receptor/protein interactions by identifying the suitable active sites in the human gamma-aminobutyric acid receptor, the gaba (a)r-beta3 homopentamer human gammaaminobutyric acid receptor, and the gaba (a)r-beta3 homopentamer protein. RESULTS: It was observed in the reaction, 3-alkyl/aryl-2-alkyl-quinazolin-4-one gave good yield as well as good quality of the product by using MW. All the synthesized compounds were subjected to grid-based molecular docking studies. The results show that compound 4t has good affinity to the active site residue of the human gamma-aminobutyric acid receptor, and the gaba (a)r-beta3 homopentamer. CONCLUSION: The Microwave irradiation for the synthesis of the title compounds offers a reduction in reaction time, operation simplicity, cleaner reaction, easy work-up and improved yields. The procedure clearly highlights the advantages of green chemistry. The data reported in this article may be helpful for the medicinal chemists who are working in this area. The protein-ligand interaction plays a significant role in structural based drug designing. In the present work, we have docked the ligand, 2, 3-disubstituted quinazolinone with the proteins that are used as the target for GABA-A receptor.

Novel Three-Finger Neurotoxins from Naja melanoleuca Cobra Venom Interact with GABAA and Nicotinic Acetylcholine Receptors.

Cobra venoms contain three-finger toxins (TFT) including α-neurotoxins efficiently binding nicotinic acetylcholine receptors (nAChRs). As shown recently, several TFTs block GABAA receptors (GABAARs) with different efficacy, an important role of the TFTs central loop in binding to these receptors being demonstrated. We supposed that the positive charge (Arg36) in this loop of α-cobratoxin may explain its high affinity to GABAAR and here studied α-neurotoxins from African cobra N. melanoleuca venom for their ability to interact with GABAARs and nAChRs. Three α-neurotoxins, close homologues of the known N. melanoleuca long neurotoxins 1 and 2, were isolated and sequenced. Their analysis on Torpedocalifornica and α7 nAChRs, as well as on acetylcholine binding proteins and on several subtypes of GABAARs, showed that all toxins interacted with the GABAAR much weaker than with the nAChR: one neurotoxin was almost as active as α-cobratoxin, while others manifested lower activity. The earlier hypothesis about the essential role of Arg36 as the determinant of high affinity to GABAAR was not confirmed, but the results obtained suggest that the toxin loop III may contribute to the efficient interaction of some long-chain neurotoxins with GABAAR. One of isolated toxins manifested different affinity to two binding sites on Torpedo nAChR.

Postsynaptic plasticity of GABAergic synapses.

The flexibility of neuronal networks is believed to rely mainly on the plasticity of excitatory synapses. However, like their excitatory counterparts, inhibitory synapses also undergo several forms of synaptic plasticity. This review examines recent advances in the understanding of the molecular mechanisms leading to postsynaptic GABAergic plasticity. Specifically, modulation of GABAA receptor (GABAAR) number at postsynaptic sites plays a key role, with the interaction of GABAARs with the scaffold protein gephyrin and other postsynaptic scaffold/regulatory proteins having particular importance. Our understanding of these molecular interactions are progressing, based on recent insights into the processes of GABAAR lateral diffusion, gephyrin dynamics, and gephyrin nanoscale organization. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Optopharmacology reveals a differential contribution of native GABAA receptors to dendritic and somatic inhibition using azogabazine.

γ-aminobutyric acid type-A receptors (GABAARs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation. To explore their physiological roles in cellular and neural network activity, it is important to understand why specific GABAAR isoforms are distributed not only to various brain regions and cell types, but also to specific areas of the membrane in individual neurons. To address this aim we have developed a novel photosensitive compound, azogabazine, that targets and reversibly inhibits GABAARs. The receptor selectivity of the compound is based on the competitive antagonist, gabazine, and photosensitivity is conferred by a photoisomerisable azobenzene group. Azogabazine can exist in either cis or trans conformations that are controlled by UV and blue light respectively, to affect receptor inhibition. We report that the trans-isomer preferentially binds and inhibits GABAAR function, whilst promotion of the cis-isomer caused unbinding of azogabazine from GABAARs. Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABAARs at somatic inhibitory synapses compared to those populating proximal dendritic zones, even though the latter displayed a higher number of synapses per unit area of membrane. Azogabazine also revealed more pronounced GABA-mediated inhibition of action potential firing in proximal dendrites compared to the soma. Overall, azogabazine is a valuable addition to the photochemical toolkit that can be used to interrogate GABAAR function and inhibition.