Genetic effects on an anesthetic‐sensitive pathway in the brain of drosophila

General anesthetics are known to inhibit the electrically induced escape response of the fruitfly through action within the brain. We examined this response and its sensitivity to anesthetics in several mutants that cause significant disruption of the mushroom body and other structures of the central brain in adult flies. Because we show here that anesthesia sensitivity is influenced by genetic background, we have used a set of congenic mutant lines. Sensitivity to halothane is normal in most of these lines, indicating that the anesthetic target is unaffected by the gross status of the central brain. Thus, for the escape response, anesthetic sensitivity is not a global feature but reflects action at a localized target. Only the mushroom body defect (mud) line showed an increased sensitivity of the escape response to halothane. Sensitivity to two other anesthetics is also perturbed in this line, albeit less dramatically so. The behavior of mud/+ heterozygotes and the comparison of brain anatomy among all the mutant lines imply that the effect of the mud mutation on anesthesia is not via gross alteration of central brain structures. The possibility that an adventitious mutation in the mud line is responsible for the effects on anesthesia is disfavored by the behavior of a heterozygote between two mud alleles. Although we do not yet know whether the mud gene encodes an anesthetic target or influences the functioning of an anesthetic‐sensitive neuron in this pathway, our work indicates that this gene regulates the effects of halothane on a circumscribed pathway. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 69–78, 2000     

Genetic effects on an anesthetic-sensitive pathway in the brain of Drosophila

General anesthetics are known to inhibit the electrically induced escape response of the fruitfly through action within the brain. We examined this response and its sensitivity to anesthetics in several mutants that cause significant disruption of the mushroom body and other structures of the central brain in adult flies. Because we show here that anesthesia sensitivity is influenced by genetic background, we have used a set of congenic mutant lines. Sensitivity to halothane is normal in most of these lines, indicating that the anesthetic target is unaffected by the gross status of the central brain. Thus, for the escape response, anesthetic sensitivity is not a global feature but reflects action at a localized target. Only the mushroom body defect (mud) line showed an increased sensitivity of the escape response to halothane. Sensitivity to two other anesthetics is also perturbed in this line, albeit less dramatically so. The behavior of mud/+ heterozygotes and the comparison of brain anatomy among all the mutant lines imply that the effect of the mud mutation on anesthesia is not via gross alteration of central brain structures. The possibility that an adventitious mutation in the mud line is responsible for the effects on anesthesia is disfavored by the behavior of a heterozygote between two mud alleles. Although we do not yet know whether the mud gene encodes an anesthetic target or influences the functioning of an anesthetic-sensitive neuron in this pathway, our work indicates that this gene regulates the effects of halothane on a circumscribed pathway.     

Determinants of the anesthetic sensitivity of neuronal nicotinic acetylcholine receptors.

Some neurotransmitter-gated ion channels are very much more sensitive to general anesthetics than others, even when they are genetically and structurally related. The most striking example of this is the extreme sensitivity of heteromeric neuronal nicotinic acetylcholine receptors to inhalational general anesthetics compared with the marked insensitivity of the closely related homomeric neuronal nicotinic receptors. Here we investigate the role of the alpha subunit in determining the anesthetic sensitivity of these receptors by using alpha(3)/alpha(7) chimeric subunits that are able to form functional homomeric receptors. By comparing the sensitivities of a number of chimeras to the inhalational agent halothane we show that the short (13 amino acids) putative extracellular loop connecting the second and third transmembrane segments is a critical determinant of anesthetic sensitivity. In addition, using site-directed mutagenesis, we show that two particular amino acids in this loop play a dominant role. When mutations are made in this loop, there is a good correlation between increasing anesthetic sensitivity and decreasing acetylcholine sensitivity. We conclude that this extracellular loop probably does not participate directly in anesthetic binding, but rather determines receptor sensitivity indirectly by playing a critical role in transducing anesthetic binding into an effect on channel gating.     

Altered anesthetic sensitivity of mice lacking ndufs4, a subunit of mitochondrial complex I

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABA(A) and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.     

Partitioning of four modern volatile general anesthetics into solvents that model buried amino acid side-chains.

Partitioning of four modern inhalational anesthetics (halothane, isoflurane, enflurane, and sevoflurane) between the gas phase and nine organic solvents that model different amino acid side-chains and lipid membrane domains was performed in an effort to define which microenvironments present in proteins and lipid bilayers might be favored. Compared to a purely aliphatic environment (hexane), the presence of an aromatic-, alcohol-, thiol- or sulfide group on the solvent improved anesthetic partitioning, by factors of 1.3-5.2 for halothane, 1.7-5.6 for isoflurane, 1.7-7.6 for enflurane, and 1.5-7.3 for sevoflurane. The most favorable solvent for halothane partitioning was ethyl methyl sulfide, a model for methionine. Enflurane and isoflurane partitioned most extensively into methanol, a model for serine, and sevoflurane into ethanol, a model for threonine. Isoflurane also partitioned favorably into ethyl methyl sulfide. The results suggest that volatile general anesthetics interact better with partly polar groups, which are present on amino acids frequently found buried in the hydrophobic core of proteins, compared to purely aliphatic side-chains. Furthermore, if an anesthetic molecule was located in a saturated region of a phospholipid bilayer membrane, there would be an energetically favorable driving force for it to move into several higher dielectric microenvironments present on membrane proteins. The results provide evidence that proteins rather than lipids are the likely targets of volatile general anesthetics in biological membranes.     

Binding of the general anesthetics propofol and halothane to human serum albumin. High resolution crystal structures

Human serum albumin (HSA) is one of the most abundant proteins in the circulatory system and plays a key role in the transport of fatty acids, metabolites, and drugs. For many drugs, binding to serum albumin is a critical determinant of their distribution and pharmacokinetics; however, there have as yet been no high resolution crystal structures published of drug-albumin complexes. Here we describe high resolution crystal structures of HSA with two of the most widely used general anesthetics, propofol and halothane. In addition, we describe a crystal structure of HSA complexed with both halothane and the fatty acid, myristate. We show that the intravenous anesthetic propofol binds at two discrete sites on HSA in preformed pockets that have been shown to accommodate fatty acids. Similarly we show that the inhalational agent halothane binds (at concentrations in the pharmacologically relevant range) at three sites that are also fatty acid binding loci. At much higher halothane concentrations, we have identified additional sites that are occupied. All of the higher affinity anesthetic binding sites are amphiphilic in nature, with both polar and apolar parts, and anesthetic binding causes only minor changes in local structure.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The 19F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the 19F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, 19F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo 19F-NMR surface coil and imaging studies.     

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.     

Ubiquitin metabolism affects cellular response to volatile anesthetics in yeast

To investigate the mechanism of action of volatile anesthetics, we are studying mutants of the yeast Saccharomyces cerevisiae that have altered sensitivity to isoflurane, a widely used clinical anesthetic. Several lines of evidence from these studies implicate a role for ubiquitin metabolism in cellular response to volatile anesthetics: (i) mutations in the ZZZ1 gene render cells resistant to isoflurane, and the ZZZ1 gene is identical to BUL1 (binds ubiquitin ligase), which appears to be involved in the ubiquitination pathway; (ii) ZZZ4, which we previously found is involved in anesthetic response, is identical to the DOA1/UFD3 gene, which was identified based on altered degradation of ubiquitinated proteins; (iii) analysis of zzz1Delta zzz4Delta double mutants suggests that these genes encode products involved in the same pathway for anesthetic response since the double mutant is no more resistant to anesthetic than either of the single mutant parents; (iv) ubiquitin ligase (MDP1/RSP5) mutants are altered in their response to isoflurane; and (v) mutants with decreased proteasome activity are resistant to isoflurane. The ZZZ1 and MDP1/RSP5 gene products appear to play important roles in determining effective anesthetic dose in yeast since increased levels of either gene increases isoflurane sensitivity whereas decreased activity decreases sensitivity. Like zzz4 strains, zzz1 mutants are resistant to all five volatile anesthetics tested, suggesting there are similarities in the mechanisms of action of a variety of volatile anesthetics in yeast and that ubiquitin metabolism affects response to all the agents examined.     

The fluorinated anesthetic halothane as a potential NMR biologic probe

Fluorinated anesthetics such as halothane preferentially partition into hydrophobic environments such as cell membranes. The ¹⁹F-NMR spectrum of halothane in a rat adenocarcinoma (with known altered lipid metabolism and membrane composition) shows an altered chemical shift pattern compared to the anesthetic in normal tissue. In eight tumor samples examined, the ¹⁹F-NMR spectra exhibit two distinct resonances, compared to a single resonance observed in normal tissues. This is explained by an enhanced or altered hydrophobic component in the tumor tissue giving rise to two discrete halothane environments. Another fluorinated anesthetic, isoflurane, shows similar behavior in distinguishing normal from diseased tissue. Given the large chemical shift range of fluorine and the inherent sensitivity of this nucleus, ¹⁹F-NMR spectra of fluorinated anesthetics can also be used to follow anesthetic degradation by the liver. The ability of fluorinated anesthetics to discriminate tissues and to monitor metabolic processes is potentially useful for in vivo ¹⁹F-NMR surface coil and imaging studies.     

Effect of Local and General Anesthetics on Interfacial Water

BackgroundWater undergoes structural change as it interfaces with hydrophilic surfaces, including the many hydrophilic surfaces within the cell. This interfacial water has become known as “Exclusion Zone (EZ) water” or “fourth-phase water” [1].MethodsWe tested the hypothesis that anesthetics diminish the amount of EZ water, and that this change may correlate with functional changes in anesthesia. By using the local anesthetics Lidocaine and Bupivacaine as well as a general inhalational anesthetic, Isoflurane, we tracked the EZ size as these anesthetics were introduced.ResultsAll three anesthetics diminished EZ size in a concentration-dependent manner at concentrations of 0.18 mM and greater for Bupivacaine, 0.85 mM and greater for Lidocaine, and 0.2% for Isoflurane. At extremely low (micromolar) concentrations, however, all three anesthetics increased EZ size.ConclusionsThe sharp increase of EZ size associated with micromolar anesthetic concentrations follows a similar pattern to induction of general anesthesia, from the excitation stage (Stage II) to the depression and overdose stages of surgical anesthesia (Stages III and IV). The results are consistent with the hypothesis that anesthetics may act on water, a fundamental organizational component common to all cells.     

Interaction of GABA and volatile anesthetics in the nematode Caenorhabditis elegans

The authors tested whether mutant strains of Caenorhabditis elegans with altered sensitivity to volatile anesthetics have altered responses to GABA or GABA-agonists. They determined the ED50s of the wild-type strain N2 and two mutant strains of C. elegans to a GABA-mimetic ivermectin (IVM) and to GABA. unc-79, a strain with increased sensitivity to halothane, was more sensitive than N2 to IVM and GABA. unc-9, a strain that suppresses the increased sensitivity of unc-79 to halothane, was less sensitive than N2 to IVM and GABA. The authors also tested whether doses of GABA or IVM and volatile anesthetics were additive in their effects on C. elegans. Halothane (2.1%) did not shift the ED50 of IVM, but was antagonistic to GABA. Enflurane (4%) was antagonistic to both IVM and GABA. However, ED50s of halothane and enflurane were unchanged in the presence of IVM (35 nM) or GABA (150 mM). The authors conclude that GABA by itself does not appear to mediate halothane or enflurane sensitivity in C. elegans.     

Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.     

Molecular dynamics simulations of C2F6 effects on gramicidin A: implications of the mechanisms of general anesthesia

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.     

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K_D) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.     

The Thermodynamics of General and Local Anesthesia

General anesthetics are known to cause depression of the freezing point of transitions in biomembranes. This is a consequence of ideal mixing of the anesthetic drugs in the membrane fluid phase and exclusion from the solid phase. Such a generic law provides physical justification of the famous Meyer-Overton rule. We show here that general anesthetics, barbiturates, and local anesthetics all display the same effect on melting transitions. Their effect is reversed by hydrostatic pressure. Thus, the thermodynamic behavior of local anesthetics is very similar to that of general anesthetics. We present a detailed thermodynamic analysis of heat capacity profiles of membranes in the presence of anesthetics. Using this analysis, we are able to describe experimentally observed calorimetric profiles and predict the anesthetic features of arbitrary molecules. In addition, we discuss the thermodynamic origin of the cutoff effect of long-chain alcohols and the additivity of the effect of general and local anesthetics.     

Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin

Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic.     

A stomatin and a degenerin interact to control anesthetic sensitivity in Caenorhabditis elegans

The mechanism of action of volatile anesthetics is unknown. In Caenorhabditis elegans, mutations in the gene unc-1 alter anesthetic sensitivity. The protein UNC-1 is a close homologue of the mammalian protein stomatin. Mammalian stomatin is thought to interact with an as-yet-unknown ion channel to control sodium flux. Using both reporter constructs and translational fusion constructs for UNC-1 and green fluorescent protein (GFP), we have shown that UNC-1 is expressed primarily within the nervous system. The expression pattern of UNC-1 is similar to that of UNC-8, a sodium channel homologue. We examined the interaction of multiple alleles of unc-1 and unc-8 with each other and with other genes affecting anesthetic sensitivity. The data indicate that the protein products of these genes interact, and that an UNC-1/UNC-8 complex is a possible anesthetic target. We propose that membrane-associated protein complexes may represent a general target for volatile anesthetics.     

Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia.     

The Thermodynamics of General and Local Anesthesia

General anesthetics are known to cause depression of the freezing point of transitions in biomembranes. This is a consequence of ideal mixing of the anesthetic drugs in the membrane fluid phase and exclusion from the solid phase. Such a generic law provides physical justification of the famous Meyer-Overton rule. We show here that general anesthetics, barbiturates, and local anesthetics all display the same effect on melting transitions. Their effect is reversed by hydrostatic pressure. Thus, the thermodynamic behavior of local anesthetics is very similar to that of general anesthetics. We present a detailed thermodynamic analysis of heat capacity profiles of membranes in the presence of anesthetics. Using this analysis, we are able to describe experimentally observed calorimetric profiles and predict the anesthetic features of arbitrary molecules. In addition, we discuss the thermodynamic origin of the cutoff effect of long-chain alcohols and the additivity of the effect of general and local anesthetics.