Radical pairs may play a role in xenon-induced general anesthesia.

Understanding the mechanisms underlying general anesthesia would be a key step towards understanding consciousness. The process of xenon-induced general anesthesia has been shown to involve electron transfer, and the potency of xenon as a general anesthetic exhibits isotopic dependence. We propose that these observations can be explained by a mechanism in which the xenon nuclear spin influences the recombination dynamics of a naturally occurring radical pair of electrons. We develop a simple model inspired by the body of work on the radical-pair mechanism in cryptochrome in the context of avian magnetoreception, and we show that our model can reproduce the observed isotopic dependence of the general anesthetic potency of xenon in mice. Our results are consistent with the idea that radical pairs of electrons with entangled spins could be important for consciousness.

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.

A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors.

The pentameric γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory ligand-gated ion channels in the central nervous system. They mediate diverse physiological functions, mutations in them are associated with mental disorders and they are the target of many drugs such as general anesthetics, anxiolytics and anti-convulsants. The five subunits of synaptic GABAARs are arranged around a central pore in the order ß-α-ß-α-γ. In the outer third of the transmembrane domain (TMD) drugs may bind to five homologous intersubunit binding sites. Etomidate binds between the pair of ß - α subunit interfaces (designated as ß+/α-) and R-mTFD-MPAB binds to an α+/ß- and an γ+/ß- subunit interface (a ß- selective ligand). Ligands that bind selectively to other homologous sites have not been characterized. We have synthesized a novel photolabel, (2,6-diisopropyl-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanol or pTFD-di-iPr-BnOH). It is a potent general anesthetic that positively modulates agonist and benzodiazepine binding. It enhances GABA-induced currents, shifting the GABA concentration-response curve to lower concentrations. Photolabeling-protection studies show that it has negligible affinity for the etomidate sites and high affinity for only one of the two R-mTFD-MPAB sites. Exploratory site-directed mutagenesis studies confirm the latter conclusions and hint that pTFD-di-iPr-BnOH may bind between the α+/ß- and α+/γ- subunits in the TMD, making it an α+ ligand. The latter α+/γ- site has not previously been implicated in ligand binding. Thus, pTFD-di-iPr-BnOH is a promising new photolabel that may open up a new pharmacology for synaptic GABAARs.

Identification, characterization, and comparison of n-alkanols and anesthetics binding to the C1b subdomain of protein kinase cα: similar function with different binding sites.

Protein kinase C (PKC) is a family of lipid-activated enzymes involved in anesthetic preconditioning signaling pathways. Previously, n-alkanols and general anesthetics have been found to activate PKC by binding to the kinase C1B subdomain. In the present study, we attempt to ascertain the molecular mechanism and interaction mode of human PKCα C1B subdomain with a variety of exogenous n-alkanols and volatile general anesthetics as well as endogenous activator phorbol ester (PE) and co-activator diacylglycerol (DG). Systematic bioinformatics analysis identifies three spatially vicinal sites on the subdomain surface to potentially accommodate small-molecule ligands, where the site 1 is a narrow, amphipathic pocket, the site 2 is a wide, flat and hydrophobic pocket, and the site 3 is a rugged, polar pocket. Further interaction modeling reveals that site 1 is the cognate binding region of natural PE activator, which can moderately simulate the kinase activity in an independent manner. The short-chain n-alkanols are speculated to also bind at the site to competitively inhibit PE-induced kinase activation. The long-chain n-alkanols and co-activator DG are found to target site 2 in a nonspecific manner, while the volatile anesthetics prefer to interact with site 3 in a specific manner. Since the site 1 is composed of two protein loops that are also shared by sites 2 and 3, binding of n-alkanols, DG and anesthetics to sites 2 and 3 can trigger a conformational displacement on the two loops, which enlarges the pocket size and changes the pocket configuration of site 1 through an allosteric mechanism, consequently enhancing kinase activation by improving PE affinity to the site.

Mechanism and Development of Modern General Anesthetics.

BACKGROUND: Before October 1846, surgery and pain were synonymous but not thereafter. Conquering pain must be one of the very few strategies that has potentially affected every human being in the world of all milestones in medicine. METHODS: This review article describes how various general anesthetics were discovered historically and how they work in the brain to induce sedative, hypnosis and immobility. Their advantages and disadvantages will also be discussed. RESULTS: Anesthesia is a relatively young field but is rapidly evolving. Currently used general anesthetics are almost invariably effective, but nagging side effects, both short (e.g., cardiac depression) and long (e.g., neurotoxicity) term, have reawakened the call for new drugs. CONCLUSION: Based on the deepening understanding of historical development and molecular targets and actions of modern anesthetics, novel general anesthetics are being investigated as potentially improved sedative-hypnotics or a key to understand the mechanism of anesthesia.

Maternal exposure to volatile anesthetics induces IL-6 in fetal brains and affects neuronal development.

Most clinically used general anesthetics have demonstrated neurotoxicity in animal studies, but the related mechanisms remain unknown. Previous studies suggest that anesthetics affect neuronal development through neuroinflammation, and significant effects of neuroinflammation on neurogenesis and neuronal disease have been shown. In the present study, we treated pregnant mice with 2% sevoflurane for 3 h at gestational day 15.5 and analyzed the expression of proinflammatory cytokines, including IL-6 and IL-17, in fetal mice brains. Sevoflurane induced IL-6 mRNA significantly, but did not upregulate IL-17. Other volatile anesthetics, including isoflurane, enflurane, and halothane, induced IL-6 mRNA in fetal brains as well as sevoflurane, but propofol did not. Sevoflurane and isoflurane showed the same effects in cultured microglia and astrocytes, but not in neurons. Because IL-6 induction in fetal brains may affect neuronal precursor cells (NPCs), numbers of NPCs in the subventricular zone were studied, revealing that maternal sevoflurane treatment significantly increases NPCs in offspring at 8 weeks after birth (p8wk). But this effect was absent in IL-6 knockout mice. Finally, behavioral experiments also revealed that maternal sevoflurane exposure causes learning impairments in p8wk offspring. These findings collectively demonstrate that maternal exposure to volatile anesthetics upregulates IL-6 in fetal mice brains, and the effects could result in long-lasting influences on neuronal development.

Towards a Comprehensive Understanding of Anesthetic Mechanisms of Action: A Decade of Discovery.

Significant progress has been made in the 21st century towards a comprehensive understanding of the mechanisms of action of general anesthetics, coincident with progress in structural biology and molecular, cellular, and systems neuroscience. This review summarizes important new findings that include target identification through structural determination of anesthetic binding sites, details of receptors and ion channels involved in neurotransmission, and the critical roles of neuronal networks in anesthetic effects on memory and consciousness. These recent developments provide a comprehensive basis for conceptualizing pharmacological control of amnesia, unconsciousness, and immobility.

Application of small-angle neutron diffraction to the localization of general anesthetics in model membranes.

We set out to explore the applicability of small-angle neutron diffraction (SAND) to the localization of biomembrane components by studying the general anesthetic n-decane in a model lipid bilayer system composed of dioleoyl-phosphocholine (DOPC). Samples in the form of planar membrane multilayers were hydrated by varied mixtures of deuterated and protonated water, and examined by the means of SAND. Neutron scattering length density (NSLD) profiles of the system were then reconstructed from the experimental data. We exploited the significantly different neutron scattering properties of hydrogen and deuterium atoms via labeling in addition to water contrast variation. Enhancing the signals from particular components of bilayer system led to a set of characteristic membrane profiles and from their comparison we localized n-decane molecules unequivocally in the bilayer's hydrocarbon chain region.

Inhibition by general anesthetic propofol of compound action potentials in the frog sciatic nerve and its chemical structure.

Although the intravenous general anesthetic propofol (2,6-diisopropylphenol) has an ability to inhibit nerve conduction, this has not been fully examined. Various agents inhibit compound action potentials (CAPs) in a manner dependent on their chemical structures. To determine propofol's chemical structure that is important in nerve conduction inhibition, we examined the effects of propofol and its related compounds on fast-conducting CAPs recorded from the frog sciatic nerve by using the air-gap method. Propofol concentration-dependently reduced the peak amplitude of the CAP with a half-maximal inhibitory concentration (IC50) value of 0.14 mM. A similar inhibition was produced by other phenols, 4-sec-butylphenol and 4-amylphenol (IC50 values: 0.33 and 0.20 mM, respectively). IC50 values for these and more phenols (4-isopropylphenol, 4-tert-butylphenol, and 4-ter-amylphenol; data published previously) were correlated with the logarithm of their octanol-water partition coefficients. A phenol having ketone group (raspberry ketone) and alcohols (3-phenyl-1-propanol and 2-phenylethylalcohol) inhibited CAPs less effectively than the above-mentioned phenols. The local anesthetic (LA) benzocaine reduced CAP peak amplitudes with an IC50 of 0.80 mM, a value larger than that of propofol. When compared with other LAs, propofol activity was close to those of ropivacaine, levobupivacaine, and pramoxine, while benzocaine activity was similar to those of cocaine and lidocaine. It is concluded that propofol inhibits nerve conduction, possibly owing to isopropyl and hydroxyl groups bound to the benzene ring of propofol and to its lipophilicity; propofol's efficacy is comparable to those of some LAs. These results could serve to develop propofol-related agents exhibiting analgesia when applied topically.

The anesthetic action of some polyhalogenated ethers-Monte Carlo method based QSAR study.

Up to this date, there has been an ongoing debate about the mode of action of general anesthetics, which have postulated many biological sites as targets for their action. However, postoperative nausea and vomiting are common problems in which inhalational agents may have a role in their development. When a mode of action is unknown, QSAR modelling is essential in drug development. To investigate the aspects of their anesthetic, QSAR models based on the Monte Carlo method were developed for a set of polyhalogenated ethers. Until now, their anesthetic action has not been completely defined, although some hypotheses have been suggested. Therefore, a QSAR model should be developed on molecular fragments that contribute to anesthetic action. QSAR models were built on the basis of optimal molecular descriptors based on the SMILES notation and local graph invariants, whereas the Monte Carlo optimization method with three random splits into the training and test set was applied for model development. Different methods, including novel Index of ideality correlation, were applied for the determination of the robustness of the model and its predictive potential. The Monte Carlo optimization process was capable of being an efficient in silico tool for building up a robust model of good statistical quality. Molecular fragments which have both positive and negative influence on anesthetic action were determined. The presented study can be useful in the search for novel anesthetics.

Electron Spin Resonance (EPR) in Drosophila and General Anesthesia.

Changes in electron spin content can be detected by X-band continuous-wave electron spin resonance (ESR, EPR) in Drosophila fruit flies without requiring the use of spin traps. The spin changes are related to cellular respiration and behave differently in anesthesia-resistant fly strains. We describe the method used in these measurements and its possible applications to the problem of the mechanism of general anesthesia.

X-Ray Crystallographic Studies for Revealing Binding Sites of General Anesthetics in Pentameric Ligand-Gated Ion Channels.

X-ray crystallography is a powerful tool in structural biology and can offer insight into structured-based understanding of general anesthetic action on various relevant molecular targets, including pentameric ligand-gated ion channels (pLGICs). In this chapter, we outline the procedures for expression and purification of pLGICs. Optimization of crystallization conditions, especially to achieve high-resolution structures of pLGICs bound with general anesthetics, is also presented. Case studies of pLGICs bound with the volatile general anesthetic isoflurane, 2-bromoethanol, and the intravenous general anesthetic ketamine are revisited.

Fluorescent Anesthetics.

Methods for using exogenous fluorophore and general anesthetic 1-aminoanthracene (1-AMA) and its photoactive derivative 1-azidoanthracene (1-AZA) are provided. 1-AMA potentiates GABAA chloride currents and immobilizes Xenopus laevis tadpoles. Cellular and tissue anesthetic distribution can be imaged for quantifying "on-pathway" and "off-pathway" targets. 1-AZA shares targets with 1-AMA and offers further optoanesthetic spatial and temporal control upon near-UV laser irradiation. Furthermore, 1-AZA adduction provides screening of possible relevant anesthetic protein targets and binding site characterization. We highlight several useful imaging and binding assays to demonstrate utility of 1-AMA and its derivative 1-AZA.

Physical Accuracy Leads to Biological Relevance: Best Practices For Simulating Ligand-Gated Ion Channels Interacting With General Anesthetics.

Efforts to detect binding modes of general anesthetics (GAs) for pentameric ligand-gated ion channels (pLGICs) are often complicated by a large number of indicated sites, as well as the challenges of ranking sites by affinity and determining which sites are occupied at clinical concentrations. Physics-based computational methods offer a powerful route for determining affinities of ligands to isolated binding sites, but preserving accuracy is essential. This chapter describes a step-by-step approach to multiple methods for identifying candidate sites and quantifying binding affinities and also discusses limitations and common pitfalls.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

Design, Synthesis, and Evaluation of Novel 2,6-Disubstituted Phenol Derivatives as General Anesthetics.

A novel series of optically active 2,6-disubstituted alkylphenols with improved anesthetic profiles compared to widely used propofol were synthesized. The incorporation of the cyclopropyl group not only increased the steric effect but also introduced stereoselective effects over their anesthetic properties. Compounds 1, 2, and 6 were selected as potential candidates for further preclinical development including studies of their water-soluble prodrugs. Clinical studies of candidate compound 6 (Haisco HSK3486) as a general anesthetic are being performed in Australia and China.

Clinical concentrations of chemically diverse general anesthetics minimally affect lipid bilayer properties.

General anesthetics have revolutionized medicine by facilitating invasive procedures, and have thus become essential drugs. However, detailed understanding of their molecular mechanisms remains elusive. A mechanism proposed over a century ago involving unspecified interactions with the lipid bilayer known as the unitary lipid-based hypothesis of anesthetic action, has been challenged by evidence for direct anesthetic interactions with a range of proteins, including transmembrane ion channels. Anesthetic concentrations in the membrane are high (10-100 mM), however, and there is no experimental evidence ruling out a role for the lipid bilayer in their ion channel effects. A recent hypothesis proposes that anesthetic-induced changes in ion channel function result from changes in bilayer lateral pressure that arise from partitioning of anesthetics into the bilayer. We examined the effects of a broad range of chemically diverse general anesthetics and related nonanesthetics on lipid bilayer properties using an established fluorescence assay that senses drug-induced changes in lipid bilayer properties. None of the compounds tested altered bilayer properties sufficiently to produce meaningful changes in ion channel function at clinically relevant concentrations. Even supra-anesthetic concentrations caused minimal bilayer effects, although much higher (toxic) concentrations of certain anesthetic agents did alter lipid bilayer properties. We conclude that general anesthetics have minimal effects on bilayer properties at clinically relevant concentrations, indicating that anesthetic effects on ion channel function are not bilayer-mediated but rather involve direct protein interactions.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.

IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing γ-aminobutyric acid type A (GABAA) receptor activation. In this article, we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABAA receptor subtypes.

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1ß3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 µM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 µM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the ß3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices.