Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem (ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone (APG)). Using cryo-electron microscopy (cryo-EM) analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1ß2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/ß subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Quetiapine Add-On Therapy May Improve Persistent Sleep Disturbances in Patients with PTSD on Stabile Combined SSRI and Benzodiazepine Combination: A One-Group Pretest-Posttest Study.

BACKGROUND: To assess potential benefits of quetiapine for persistent sleep disturbances in patients with posttraumatic stress disorder (PTSD) on stable combined SSRI and benzodiazepine therapy, who previously failed to respond to various benzodiazepine and non-benzodiazepine hypnotic adjuvant treatment as well as to first-generation antipsychotic add-on treatment. SUBJECTS AND METHODS: Fifty-two male PTSD outpatients on stable combination treatment with SSRI and benzodiazepines, with persistent sleep disturbances not responding to prescription of zolpidem, flurazepam, nitrazepam, promazine, and levopromazine, were assessed for sleep disturbances improvements after prescription of quetiapine in the evening. Each patient met both ICD-10 and DSM-IV criteria for PTSD. Psychiatric comorbidity and premorbidity were excluded using the Mini-International Neuropsychiatric Interview (MINI). Improvement on the CAPS recurrent distressing dream item, reduction in the amount of time needed to fall asleep, prolongation of sleep duration, and reduction in average number of arousals per night in the last 7 days before the assessment period were used as efficacy measures. RESULTS: All sleep-related parameters improved significantly at the end of a five-week follow-up: sleep duration increased by one hour (p<0.001), sleep latency decreased by 52.5 minutes (p<0.001), median number of arousals per night decreased from two to one (p<0.001), CAPS recurrent distressing dream item median decreased from five to four (p<0.001), and the number of patients dissatisfied with their sleep quality and quantity decreased from 45 to two (p<0.001). CONCLUSION: Quetiapine prescribed in the evening may be successful therapy for persistent sleep disturbances in patients with PTSD and generally good response to an SSRI and benzodiazepine combination, who previously failed to respond to some of the usual hypnotic medication or addition of first-generation antipsychotics: zolpidem, flurazepam, nitrazepam, promazine, and levopromazine.

Comparison of kinetic and pharmacological profiles of recombinant α1γ2L and α1ß2γ2L GABAA receptors - A clue to the role of intersubunit interactions.

The fastest inhibitory mechanism in the CNS is mediated by ionotropic GABAA receptors and it is known that subunit composition critically determines their properties. While a typical GABAA receptor consists of two α, two ß and one γ/δ subunit, there are some exceptions, e.g. αß receptors. Functional α1γ2 GABAA receptors can be expressed in recombinant model (Verdoorn et al., 1990) and although their role remains unknown, it seems appealing to extend their characterization to further explore the structure-function relationship of GABAA receptors. Intriguingly, this receptor is lacking canonical GABA binding sites but it can be activated by GABA and dose-response relationships for α1ß2γ2L and α1γ2L receptors overlap. Deactivation kinetics was similar for both receptors but the percentage of the fast component was smaller in the case of α1γ2L receptors and, consequently, the mean deactivation time constant was slower. The rate and extent of macroscopic desensitization were smaller in the case of α1γ2L receptors but they showed slower recovery. Both receptor types had a similar proton sensitivity showing only subtle but significant differences in pH effects on deactivation. Flurazepam exerted a similar effect on both receptors but the rapid deactivation components were differently affected and an opposite effect was observed on desensitization extent. Rebound currents evoked by pentobarbital were undistinguishable for both receptor types. Taking altogether, although some significant differences were found, α1ß2γ2L and α1γ2L receptors showed unforeseen similarity. We propose that functioning of GABAA receptors might rely on subunit-subunit cooperative interactions to a larger extent than believed so far.

Role of interleukin-1 receptor signaling in the behavioral effects of ethanol and benzodiazepines.

Gene expression studies identified the interleukin-1 receptor type I (IL-1R1) as part of a pathway associated with a genetic predisposition to high alcohol consumption, and lack of the endogenous IL-1 receptor antagonist (IL-1ra) strongly reduced ethanol intake in mice. Here, we compared ethanol-mediated behaviors in mice lacking Il1rn or Il1r1. Deletion of Il1rn (the gene encoding IL-1ra) increases sensitivity to the sedative/hypnotic effects of ethanol and flurazepam and reduces severity of acute ethanol withdrawal. Conversely, deletion of Il1r1 (the gene encoding the IL-1 receptor type I, IL-1R1) reduces sensitivity to the sedative effects of ethanol and flurazepam and increases the severity of acute ethanol withdrawal. The sedative effects of ketamine and pentobarbital were not altered in the knockout (KO) strains. Ethanol intake and preference were not changed in mice lacking Il1r1 in three different tests of ethanol consumption. Recovery from ethanol-induced motor incoordination was only altered in female mice lacking Il1r1. Mice lacking Il1rn (but not Il1r1) showed increased ethanol clearance and decreased ethanol-induced conditioned taste aversion. The increased ethanol- and flurazepam-induced sedation in Il1rn KO mice was decreased by administration of IL-1ra (Kineret), and pre-treatment with Kineret also restored the severity of acute ethanol withdrawal. Ethanol-induced sedation and withdrawal severity were changed in opposite directions in the null mutants, indicating that these responses are likely regulated by IL-1R1 signaling, whereas ethanol intake and preference do not appear to be solely regulated by this pathway.

Synaptic GABAA receptor clustering without the γ2 subunit.

Rapid activation of postsynaptic GABAA receptors (GABAARs) is crucial in many neuronal functions, including the synchronization of neuronal ensembles and controlling the precise timing of action potentials. Although the γ2 subunit is believed to be essential for the postsynaptic clustering of GABAARs, synaptic currents have been detected in neurons obtained from γ2(-/-) mice. To determine the role of the γ2 subunit in synaptic GABAAR enrichment, we performed a spatially and temporally controlled γ2 subunit deletion by injecting Cre-expressing viral vectors into the neocortex of GABAARγ2(77I)lox mice. Whole-cell recordings revealed the presence of miniature IPSCs in Cre(+) layer 2/3 pyramidal cells (PCs) with unchanged amplitudes and rise times, but significantly prolonged decays. Such slowly decaying currents could be evoked in PCs by action potentials in presynaptic fast-spiking interneurons. Freeze-fracture replica immunogold labeling revealed the presence of the α1 and ß3 subunits in perisomatic synapses of cells that lack the γ2 subunit. Miniature IPSCs in Cre(+) PCs were insensitive to low concentrations of flurazepam, providing a pharmacological confirmation of the lack of the γ2 subunit. Receptors assembled from only αß subunits were unlikely because Zn(2+) did not block the synaptic currents. Pharmacological experiments indicated that the αßγ3 receptor, rather than the αßδ, αßε, or αßγ1 receptors, was responsible for the slowly decaying IPSCs. Our data demonstrate the presence of IPSCs and the synaptic enrichment of the α1 and ß3 subunits and suggest that the γ3 subunit is the most likely candidate for clustering GABAARs at synapses in the absence of the γ2 subunit.

Implication of NGF and endocannabinoid signaling in the mechanism of action of sesamol: a multi-target natural compound with therapeutic potential.

RATIONALE: Sesamol, a natural compound with anti-inflammatory, antioxidant and neuroprotective properties, has shown promising antidepressant-like effects. However, its molecular target(s) have not been well defined, which merits further investigation. OBJECTIVES: Based on the interaction between the neurotrophin and endocannabinoid (eCB) systems and their contribution to emotional reactivity and antidepressant action, we aimed to investigate the involvement of nerve growth factor (NGF) and eCB signalling in the mechanism of action of sesamol. METHODS: Following acute and 4-week intraperitoneal (i.p.) administration of sesamol (40, 80 and 100 mg/kg), the classical antidepressant amitriptyline (2.5, 5 and 10 mg/kg) or the benzodiazepine flurazepam (5, 10 and 20 mg/kg), brain regional levels of NGF and eCB contents were quantified in rats by Bio-Rad protein assay and isotope-dilution liquid chromatography/mass spectrometry, respectively. In the case of any significant change, the cannabinoid CB1 and CB2 receptor antagonists (AM251 and SR144528) were administered i.p. 30 min prior to the injection of sesamol, amitriptyline or flurazepam. RESULTS: Following the chronic treatment, sesamol, similar to amitriptyline, resulted in the sustained elevation of NGF and eCB contents in dose-dependent and brain region-specific fashion. Neither acute nor chronic treatment with flurazepam altered brain NGF or eCB contents. Pretreatment with 3 mg/kg AM251, but not SR144528, prevented the elevation of NGF protein levels. AM251 exerted no effect by itself. CONCLUSIONS: Sesamol, similar to amitriptyline, is able to affect brain NGF and eCB signalling under the regulatory drive of the CB1 receptors.

Gender differences in highway driving performance after administration of sleep medication: a review of the literature.

OBJECTIVES: It is generally assumed that there are minimal gender differences in the safety and efficacy of central nervous system drugs, as is evidenced by men and women receiving the same drug dosage. There is, however, evidence that drugs may have a differential effect on performance in men and women, given reported differences in pharmacokinetics as well as the presence or absence and severity of adverse effects. It is especially important to verify whether gender differences in performance exist in case of activities that have potentially dangerous outcomes such as driving a car. This review summarizes the current scientific evidence on gender differences in driving performance after treatment with hypnotic drugs. METHODS: A literature search was conducted to obtain all studies that conducted on-road driving tests to examine the effects hypnotic drugs on driving. Cross-references were checked and technical reports and raw data were obtained, if possible. RESULTS: Fourteen studies were evaluated. Many studies did not allow analyses of gender effects because only women were included. Others did not report data on gender analyses. Technical reports and additional data analyses revealed significant gender differences in driving performance the morning following bedtime administration of flurazepam (30 mg) and after middle-of-the-night administration of zolpidem (10 mg). No significant gender differences were found for ramelteon (8 mg), lormetazepam (1 and 2 mg), zaleplon (10 and 20 mg), and zopiclone (7.5 mg). CONCLUSIONS: Although the available data are limited, the results show that significant gender differences have been found for some drugs but not others. Therefore, in the future more research is needed to reveal potential gender differences and to determine what mediates them.

Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in α1 knock-in mice and three in the α2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the α2 mutants, and about 50% in the α1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between α2 mutants and wild-type, but they were enhanced for α1 knock-in mice. These results indicate that introduction of these mutations in the α2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the α1 subunit produce more pronounced changes, especially in GABAergic function.

Disulphide trapping of the GABA(A) receptor reveals the importance of the coupling interface in the action of benzodiazepines.

BACKGROUND AND SIGNIFICANCE: Although the functional effects of benzodiazepines (BZDs) on GABA(A) receptors have been well characterized, the structural mechanism by which these modulators alter activation of the receptor by GABA is still undefined. EXPERIMENTAL APPROACH: We used disulphide trapping between engineered cysteines to probe BZD-induced conformational changes within the γ2 subunit and at the α1/γ2 coupling interface (Loops 2, 7 and 9) of α1ß2γ2 GABA(A) receptors. KEY RESULTS: Crosslinking γ2 Loop 9 to γ2ß-strand 9 (via γ2 S195C/F203C and γ2 S187C/L206C) significantly decreased maximum potentiation by flurazepam, suggesting that modulation of GABA-induced current (I(GABA)) by flurazepam involves movements of γ2 Loop 9 relative to γ2ß-strand 9. In contrast, tethering γ2ß-strand 9 to the γ2 pre-M1 region (via γ2S202C/S230C) significantly enhanced potentiation by both flurazepam and zolpidem, indicating γ2S202C/S230C trapped the receptor in a more favourable conformation for positive modulation by BZDs. Intersubunit disulphide bonds formed at the α/γ coupling interface between α1 Loop 2 and γ2Loop 9 (α1D56C/γ2L198C) prevented flurazepam and zolpidem from efficiently modulating I(GABA) . Disulphide trapping α1 Loop 2 (α1D56C) to γ2ß-strand 1 (γ2P64C) decreased maximal I(GABA) as well as flurazepam potentiation. None of the disulphide bonds affected the ability of the negative modulator, 3-carbomethoxy-4-ethyl-6,7-dimethoxy-ß-carboline (DMCM), to inhibit I(GABA) . CONCLUSIONS AND IMPLICATIONS: Positive modulation of GABA(A) receptors by BZDs requires reorganization of the loops in the α1/γ2 coupling interface. BZD-induced movements at the α/γ coupling interface likely synergize with rearrangements induced by GABA binding at the ß/α subunit interfaces to enhance channel activation by GABA.

Calcium/calmodulin-dependent protein kinase II mediates hippocampal glutamatergic plasticity during benzodiazepine withdrawal.

Benzodiazepine withdrawal anxiety is associated with potentiation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) currents in hippocampal CA1 pyramidal neurons attributable to increased synaptic incorporation of GluA1-containing AMPARs. The contribution of calcium/calmodulin-dependent protein kinase II (CaMKII) to enhanced glutamatergic synaptic strength during withdrawal from 1-week oral flurazepam (FZP) administration was further examined in hippocampal slices. As earlier reported, AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, along with increased single-channel conductance in neurons from 2-day rats, estimated by non-stationary noise analysis. Input-output curve slope was increased without a change in paired-pulse facilitation, suggesting increased AMPAR postsynaptic efficacy rather than altered glutamate release. The increased mEPSC amplitude and AMPAR conductance were related to CaMKII activity, as intracellular inclusion of CaMKIINtide or autocamtide-2-related inhibitory peptide, but not scrambled peptide, prevented both AMPAR amplitude and conductance changes. mEPSC inhibition by 1-naphthyl acetyl spermine and the negative shift in rectification index at both withdrawal time points were consistent with functional incorporation of GluA2-lacking AMPARs. GluA1 but not GluA2 or GluA3 levels were increased in immunoblots of postsynaptic density (PSD)-enriched subcellular fractions of CA1 minislices from 1-day FZP-withdrawn rats, when mEPSC amplitude, but not conductance, was increased. Both GluA1 expression levels and CaMKII alpha-mediated GluA1 Ser(831) phosphorylation were increased in PSD-subfractions from 2-day FZP-withdrawn rats. As phospho-Thr(286)CaMKII alpha was unchanged, CaMKII alpha may be activated through an alternative signaling pathway. Synaptic insertion and subsequent CaMKII alpha-mediated Ser(831) phosphorylation of GluA1 homomers contribute to benzodiazepine withdrawal-induced AMPAR potentiation and may represent an important hippocampal pathway mediating both drug-induced and activity-dependent plasticity.

[Drugs and the mechanism for reversing the tolerance of flurazepan in rats].

OBJECTIVE: Benzodiazepines (BDZ) have many effects on various kinds of epilepsies, but long-term treatment with BDZ often leads to drug tolerance. This study aimed to seek drugs which can reverse the tolerance of flurazepam (FZP), and to explore the role of neuropeptide Y (NPY) in the reversal effect. METHODS: A rat model of anticonvulsant tolerance to FZP was prepared. The rats with FZP tolerance were randomly assigned to seven groups: FZP-tolerance, and nifedipine, levetiracetam, topiramate, flumazenil, L-NAME and pyridoxamine treatment groups. The tolerance to FZP was evaluated through pentylenetetrazol (PTZ) infusion into a tail vein. The latency to onset of clonic seizure and the PTZ threshold were recorded. The mRNA of NPY receptor Y2 in the hippocampus was determined by RT-PCR, and the distribution of NPY in the hippocampus was examined by immunohistochemistry. RESULTS: In comparison with the blank control group, the average latency to the onset of clonic seizure was shortened, the average PTZ threshold decreased and the expression of NYT and NPY receptor Y2 mRNA decreased significantly in the FZP-tolerance group (p<0.01). In comparison with the FZP-tolerance group, the average latency to onset of clonic seizure was prolonged by 2 times and the average PTZ threshold doubled in the topiramate treatment group. The average latency to onset of clonic seizure was prolonged by 1 time and the average PTZ threshold increased 1 time in the nifedipine, the levetiracetam and the flumazenil treatment groups. The mRNA expression of NPY receptor Y2 increased by 1 or 2 times in the flumazenil, the nifedipine and the topiramate treatment groups when compared with the FZP-tolerance group. CONCLUSIONS: Nifedipine, levetiracetam, topiramate and flumazenil can reverse the anticonvulsant tolerance to flurazepam. NPY may play a role in mediating the reversal effect.

Genetically inbred Balb/C mice are more sensitive to an effect of flurazepam and more resistant to an effect of stress than a genetically outbred mouse strain.

The inbred Balb/c mouse strain was more sensitive than the outbred NIH Swiss mouse to flurazepam's ability to antagonize electrically precipitated seizures. In prior work, a reduction in flurazepam's antiseizure efficacy was not observed 24h after forcing Balb/c mice to swim for up to 10 min in ambient temperature water. Thus, we wondered if a stress-induced reduction would be observed after forcing mice to swim for up to 10 min in cold (6 degrees C) water, a more severe stress. The current data show that 24 h after exposure to this stress, the ability of flurazepam to raise the threshold voltage for the elicitation of tonic hindlimb extension in the Balb/c mouse strain was reduced. The genetically inbred Balb/c mouse strain is emerging as an interesting animal model in which to study interactions of stress and genetic factors that affect endogenous neurotransmission mediated by l-glutamate and GABA at the NMDA and GABA(A) receptor complexes, respectively.

An epigenetic intervention interacts with genetic strain differences to modulate the stress-induced reduction of flurazepam's antiseizure efficacy in the mouse.

Stress induces changes in the endogenous tone of both GABA and NMDA receptor-mediated neurotransmission in the intact mouse. Because changes are observed 24 h after stress, epigenetically-regulated alterations in gene expression may mediate these effects. In earlier work, sodium butyrate, a centrally-active histone deacetylase inhibitor that promotes gene expression, was shown to modulate the stress-induced reduction of the ability of MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, to antagonize electrically-precipitated seizures. In the current study, we extended this work to look at sodium butyrate's modulatory effect on stress-induced changes in the antiseizure efficacy of flurazepam, a benzodiazepine receptor agonist, in two strains of mice. Epigenetic mechanisms, genetic strain differences and a standard stress interacted to alter flurazepam's antiseizure efficacy. These data support examination and development of epigenetic treatment strategies.

17beta-estradiol affects GABAergic transmission in developing hippocampus.

Estrogens are potent modulators of the nervous system. In particular, 17beta-estradiol was shown to affect GABAergic synaptic transmission in hippocampus of adult animals in vivo but much less is known on the impact of this hormone on the GABAergic system in the developing brains. We have recently shown that phasic and tonic GABAergic transmissions are strongly modulated upon long-term treatment with exogenous 17beta-estradiol in hippocampal neurons developing in vitro. To check for the long-term estrogen effect in a more physiological developmental model, we have investigated the GABAergic transmission in developing brains of P7-P40 animals, injected daily with 17beta-estradiol. We have found that such a treatment clearly increased GABAergic mIPSC frequency and amplitude while the onset and decay of mIPSCs were shortened. These effects were statistically significant in the youngest considered age group (P7-P13) with a tendency to disappear in older animals. Long-term treatment with estradiol did not change the susceptibility of mIPSC amplitude to upregulation by flurazepam while mIPSC decay was prolonged by this drug to a larger extent in 17beta-estradiol-treated animals. 17beta-estradiol strongly upregulated GABAergic tonic current but again this effect was restricted to the youngest group of animals. We conclude that 17beta-estradiol strongly modulates the GABAergic synaptic transmission but this effect critically depends on the animal age being the most prominent in youngest animals.

Chronic benzodiazepine-induced reduction in GABA(A) receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection.

One week oral flurazepam (FZP) administration in rats results in reduced GABA(A) receptor-mediated synaptic transmission in CA1 pyramidal neurons associated with benzodiazepine tolerance in vivo and in vitro. Since voltage-gated calcium channel (VGCC) current density is enhanced twofold during chronic FZP treatment, the role of L-type VGCCs in regulating benzodiazepine-induced changes in CA1 neuron GABA(A) receptor-mediated function was evaluated. Nimodipine (10 mg/kg, i.p.) or vehicle (0.5% Tween 80, 2 ml/kg) was injected 1 day after ending FZP treatment and 24 h prior to hippocampal slice preparation for measurement of mIPSC characteristics and in vitro tolerance to zolpidem. The reduction in GABA(A) receptor-mediated mIPSC amplitude and estimated unitary channel conductance measured 2 days after drug removal was no longer observed following prior nimodipine injection. However, the single nimodipine injection failed to prevent in vitro tolerance to zolpidem's ability to prolong mIPSC decay in FZP-treated neurons, suggesting multiple mechanisms may be involved in regulating GABA(A) receptor-mediated synaptic transmission following chronic FZP administration. As reported previously in recombinant receptors, nimodipine inhibited synaptic GABA(A) receptor currents only at high concentrations (>30 muM), significantly greater than attained in vivo (1 muM) 45 min after a single antagonist injection. Thus, the effects of nimodipine were unlikely to be related to direct effects on GABA(A) receptors. As with nimodipine injection, buffering intracellular free [Ca(2+)] with BAPTA similarly prevented the effects on GABA(A) receptor-mediated synaptic transmission, suggesting intracellular Ca(2+) homeostasis is important to maintain GABA(A) receptor function. The findings further support a role for activation of L-type VGCCs, and perhaps other Ca(2+)-mediated signaling pathways, in the modulation of GABA(A) receptor synaptic function following chronic benzodiazepine administration, independent of modulation of the allosteric interactions between benzodiazepine and GABA binding sites.

K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

Individually monitoring ligand-induced changes in the structure of the GABAA receptor at benzodiazepine binding site and non-binding-site interfaces.

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (alpha/gamma interface) and at aligned positions in the alpha/beta interface. alpha(1)His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of alpha(1)H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of alpha(1)H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of alpha(1)H101C at the alpha/beta interface was significantly slower than modification of alpha(1)H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the alpha/gamma and alpha/beta interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near alpha(1)H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.

Flurazepam effect on GABAergic currents depends on extracellular pH.

BACKGROUND AND PURPOSE: Benzodiazepines (BDZs) are widely used in clinical practice and are known as positive modulators of GABAergic currents. BDZs increase binding affinity and recently they were found to affect GABA(A) receptor gating, including desensitization. Binding and desensitization are also strongly modulated by extracellular pH, a factor that may be severely altered in a pathological brain. It is thus of interest to examine the combined action of BDZ and protons. EXPERIMENTAL APPROACH: Pharmacokinetic analysis was based on patch clamp recordings of miniature IPSCs (mIPSCs) and current responses to GABA applications in rat cultured hippocampal neurons. High temporal resolution of currents evoked by exogenous GABA was achieved by using an ultrafast perfusion system (exchange time ca. 80 micros). KEY RESULTS: At acidic pH, flurazepam produced a stronger enhancement of mIPSC amplitudes than at physiological pH. At low GABA concentrations, flurazepam markedly enhanced current amplitudes both at normal and acidic pH, but at the latter, the relative effect was larger. In contrast, at saturating GABA concentrations, flurazepam reduced current amplitudes at both pH 7.2 and 6.0. The slowing of deactivation kinetics by flurazepam decreased with GABA concentration, but at pH 6.0, this trend was shifted toward a higher GABA concentration. CONCLUSIONS AND IMPLICATIONS: Acidification of extracellular medium may significantly affect the susceptibility of phasic and tonic components of GABAergic currents to modulation by BDZs. Quantitative analysis and model simulations indicate that protons and flurazepam additively affect binding and desensitization of GABA(A) receptors.

Flurazepam inhibits the P-glycoprotein transport function: an insight to revert multidrug-resistance phenotype.

P-glycoprotein mediated drug transport may lead to a multidrug resistance phenotype often associated with a poor response to the successful treatment of a variety of human disorders. Several agents have been found to modulate P-glycoprotein drug resistance, most probably by blocking its transport function. The aim of this study was to examine the effects of some benzodiazepines (bromazepam, chlordiazepoxide, diazepam and flurazepam) able to bind to P-glycoprotein in proteoliposomes on its transport function and ATPase activity in the human cancer cell line, KB-V1. The toxicity of the benzodiazepines drugs towards KB-V1 cells was first evaluated and the non toxic drugs concentrations were used to assess the drug efflux and the ATPase activity. Using the flow cytometry approach, the accumulation and efflux of daunorubicin were followed by measuring the daunorubicin associated geometric mean fluorescence intensity. Vanadate was employed as a comparative inhibitory compound. Flurazepam was able to inhibit the daunorubicin efflux in 80%. ATPase activity determined by a colorimetric assay revealed that flurazepam inhibits the P-glycoprotein enzymatic activity, indicating coupling between drug transport and ATP hydrolysis. Bromazepam, chlordiazepoxide and diazepam behaved as activators of the P-glycoprotein ATPase activity, suggesting a role as transported substrates and did not interfere in the daunorubicin transport.

GABA transient sets the susceptibility of mIPSCs to modulation by benzodiazepine receptor agonists in rat hippocampal neurons.

Benzodiazepines (BDZs) are known to increase the amplitude and duration of IPSCs. Moreover, at low [GABA], BDZs strongly enhance GABAergic currents suggesting the up-regulation of agonist binding while their action on gating remains a matter of debate. In the present study we have examined the impact of flurazepam and zolpidem on mIPSCs by investigating their effects on GABA(A)R binding and gating and by considering dynamic conditions of synaptic receptor activation. Flurazepam and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both compounds strongly enhanced responses to low [GABA] but, surprisingly, decreased the currents evoked by saturating or half-saturating [GABA]. Analysis of current responses to ultrafast GABA applications indicated that these compounds enhanced binding and desensitization of GABA(A) receptors. Flurazepam and zolpidem markedly prolonged deactivation of responses to low [GABA] but had almost no effect on deactivation at saturating or half-saturating [GABA]. Moreover, at low [GABA], flurazepam enhanced desensitization-deactivation coupling but zolpidem did not. Recordings of responses to half-saturating [GABA] applications revealed that appropriate timing of agonist exposure was sufficient to reproduce either a decrease or enhancement of currents by flurazepam or zolpidem. Recordings of currents mediated by recombinant ('synaptic') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.