Mapping the contribution of β3-containing GABA(A )receptors to volatile and intravenous general anesthetic actions

BACKGROUND: Agents belonging to diverse chemical classes are used clinically as general anesthetics. The molecular targets mediating their actions are however still only poorly defined. Both chemical diversity and substantial differences in the clinical actions of general anesthetics suggest that general anesthetic agents may have distinct pharmacological targets. It was demonstrated previously that the immobilizing action of etomidate and propofol is completely, and the immobilizing action of isoflurane partly mediated, by β3-containing GABA(A )receptors. This was determined by using the β3(N265M) mice, which carry a point mutation known to decrease the actions of general anesthetics at recombinant GABA(A )receptors. In this communication, we analyzed the contribution of β3-containing GABA(A )receptors to the pharmacological actions of isoflurane, etomidate and propofol by means of β3(N265M) mice. RESULTS: Isoflurane decreased core body temperature and heart rate to a smaller degree in β3(N265M) mice than in wild type mice, indicating a minor but significant role of β3-containing GABA(A )receptors in these actions. Prolonged time intervals in the ECG and increased heart rate variability were indistinguishable between genotypes, suggesting no involvement of β3-containing GABA(A )receptors. The anterograde amnesic action of propofol was indistinguishable in β3(N265M) and wild type mice, suggesting that it is independent of β3-containing GABA(A )receptors. The increase of heart rate variability and prolongation of ECG intervals by etomidate and propofol were also less pronounced in β3(N265M) mice than in wild type mice, pointing to a limited involvement of β3-containing GABA(A )receptors in these actions. The lack of etomidate- and propofol-induced immobilization in β3(N265M) mice was also observed in congenic 129X1/SvJ and C57BL/6J backgrounds, indicating that this phenotype is stable across different backgrounds. CONCLUSION: Our results provide evidence for a defined role of β3-containing GABA(A )receptors in mediating some, but not all, of the actions of general anesthetics, and confirm the multisite model of general anesthetic action. This pharmacological separation of anesthetic endpoints also suggests that subtype-selective substances with an improved side-effect profile may be developed

Molecular modeling of a tandem two pore domain potassium channel reveals a putative binding Site for general anesthetics

[Image: see text] Anesthetics are thought to mediate a portion of their activity via binding to and modulation of potassium channels. In particular, tandem pore potassium channels (K2P) are transmembrane ion channels whose current is modulated by the presence of general anesthetics and whose genetic absence has been shown to confer a level of anesthetic resistance. While the exact molecular structure of all K2P forms remains unknown, significant progress has been made toward understanding their structure and interactions with anesthetics via the methods of molecular modeling, coupled with the recently released higher resolution structures of homologous potassium channels to act as templates. Such models reveal the convergence of amino acid regions that are known to modulate anesthetic activity onto a common three- dimensional cavity that forms a putative anesthetic binding site. The model successfully predicts additional important residues that are also involved in the putative binding site as validated by the results of suggested experimental mutations. Such a model can now be used to further predict other amino acid residues that may be intimately involved in the target-based structure–activity relationships that are necessary for anesthetic binding

Protective Effects of Walnut Extract Against Amyloid Beta Peptide-Induced Cell Death and Oxidative Stress in PC12 Cells

Amyloid beta-protein (Aβ) is the major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Recently, considerable attention has been focused on dietary antioxidants that are able to scavenge reactive oxygen species (ROS), thereby offering protection against oxidative stress. Walnuts are rich in components that have anti-oxidant and anti-inflammatory properties. The inhibition of in vitro fibrillization of synthetic Aβ, and solubilization of preformed fibrillar Aβ by walnut extract was previously reported. The present study was designed to investigate whether walnut extract can protect against Aβ-induced oxidative damage and cytotoxicity. The effect of walnut extract on Aβ-induced cellular damage, ROS generation and apoptosis in PC12 pheochromocytoma cells was studied. Walnut extract reduced Aβ-mediated cell death assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, and release of lactate dehydrogenase (membrane damage), DNA damage (apoptosis) and generation of ROS in a concentration-dependent manner. These results suggest that walnut extract can counteract Aβ-induced oxidative stress and associated cell death

GABAA receptors as molecular targets of general anesthetics: identification of binding sites provides clues to allosteric modulation

PurposeThe purpose of this review is to summarize current knowledge of detailed biochemical evidence for the role of γ-aminobutyric acid type A receptors (GABA(A)-Rs) in the mechanisms of general anesthesia.Principal findingsWith the knowledge that all general anesthetics positively modulate GABA(A)-R-mediated inhibitory transmission, site-directed mutagenesis comparing sequences of GABA(A)-R subunits of varying sensitivity led to identification of amino acid residues in the transmembrane domain that are critical for the drug actions in vitro. Using a photo incorporable analogue of the general anesthetic, R(+)etomidate, we identified two transmembrane amino acids that were affinity labelled in purified bovine brain GABA(A)-R. Homology protein structural modelling positions these two residues, αM1-11' and βM3-4', close to each other in a single type of intersubunit etomidate binding pocket at the β/α interface. This position would be appropriate for modulation of agonist channel gating. Overall, available information suggests that these two etomidate binding residues are allosterically coupled to sites of action of steroids, barbiturates, volatile agents, and propofol, but not alcohols. Residue α/βM2-15' is probably not a binding site but allosterically coupled to action of volatile agents, alcohols, and intravenous agents, and α/βM1-(-2') is coupled to action of intravenous agents.ConclusionsEstablishment of a coherent and consistent structural model of the GABA(A)-R lends support to the conclusion that general anesthetics can modulate function by binding to appropriate domains on the protein. Genetic engineering of mice with mutation in some of these GABA(A)-R residues are insensitive to general anesthetics in vivo, suggesting that further analysis of these domains could lead to development of more potent and specific drugs

Recognition of Anesthetic Barbiturates by a Protein Binding Site: A High Resolution Structural Analysis

Barbiturates potentiate GABA actions at the GABAA receptor and act as central nervous system depressants that can induce effects ranging from sedation to general anesthesia. No structural information has been available about how barbiturates are recognized by their protein targets. For this reason, we tested whether these drugs were able to bind specifically to horse spleen apoferritin, a model protein that has previously been shown to bind many anesthetic agents with affinities that are closely correlated with anesthetic potency. Thiopental, pentobarbital, and phenobarbital were all found to bind to apoferritin with affinities ranging from 10–500 µM, approximately matching the concentrations required to produce anesthetic and GABAergic responses. X-ray crystal structures were determined for the complexes of apoferritin with thiopental and pentobarbital at resolutions of 1.9 and 2.0 Å, respectively. These structures reveal that the barbiturates bind to a cavity in the apoferritin shell that also binds haloalkanes, halogenated ethers, and propofol. Unlike these other general anesthetics, however, which rely entirely upon van der Waals interactions and the hydrophobic effect for recognition, the barbiturates are recognized in the apoferritin site using a mixture of both polar and nonpolar interactions. These results suggest that any protein binding site that is able to recognize and respond to the chemically and structurally diverse set of compounds used as general anesthetics is likely to include a versatile mixture of both polar and hydrophobic elements

The neurobiology of mouse models syntenic to human chromosome 15q

Autism is a neurodevelopmental disorder that manifests in childhood as social behavioral abnormalities, such as abnormal social interaction, impaired communication, and restricted interest or behavior. Of the known causes of autism, duplication of human chromosome 15q11–q13 is the most frequently associated cytogenetic abnormality. Chromosome 15q11–q13 is also known to include imprinting genes. In terms of neuroscience, it contains interesting genes such as Necdin, Ube3a, and a cluster of GABAA subunits as well as huge clusters of non-coding RNAs (small nucleolar RNAs, snoRNAs). Phenotypic analyses of mice genetically or chromosomally engineered for each gene or their clusters on a region of mouse chromosome seven syntenic to human 15q11–q13 indicate that this region may be involved in social behavior, serotonin metabolism, and weight control. Further studies using these models will provide important clues to the pathophysiology of autism. This review overviews phenotypes of mouse models of genes in 15q11–q13 and their relationships to autism