Molecular Mechanism Of General Anesthesia Research Articles

Understanding the Molecular Mechanism of Anesthesia: Effect of General Anesthetics and Structurally Similar Non-Anesthetics on the Properties of Lipid Membranes.

General anesthesia can be caused by various, chemically very different molecules, while several other molecules, many of which are structurally rather similar to them, do not exhibit anesthetic effects at all. To understand the origin of this difference and shed some light on the molecular mechanism of general anesthesia, we report here molecular dynamics simulations of the neat dipalmitoylphosphatidylcholine (DPPC) membrane as well as DPPC membranes containing the anesthetics diethyl ether and chloroform and the structurally similar non-anesthetics n-pentane and carbon tetrachloride, respectively. To also account for the pressure reversal of anesthesia, these simulations are performed both at 1 bar and at 600 bar. Our results indicate that all solutes considered prefer to stay both in the middle of the membrane and close to the boundary of the hydrocarbon domain, at the vicinity of the crowded region of the polar headgroups. However, this latter preference is considerably stronger for the (weakly polar) anesthetics than for the (apolar) non-anesthetics. Anesthetics staying in this outer preferred position increase the lateral separation between the lipid molecules, giving rise to a decrease of the lateral density. The lower lateral density leads to an increased mobility of the DPPC molecules, a decreased order of their tails, an increase of the free volume around this outer preferred position, and a decrease of the lateral pressure at the hydrocarbon side of the apolar/polar interface, a change that might well be in a causal relation with the occurrence of the anesthetic effect. All these changes are clearly reverted by the increase of pressure. Furthermore, non-anesthetics occur in this outer preferred position in a considerably smaller concentration and hence either induce such changes in a much weaker form or do not induce them at all.

Effect of xenon, an apolar general anaesthetic on the properties of the DPPC bilayer

The effect of xenon, a general anaesthetic, on the properties of the fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer is investigated by molecular dynamics simulations with two different force fields. Considering the well-known pressure reversal of the anaesthetic effect, we are looking for such changes that are reverted by the increase of the pressure, and hence can be relevant in explaining the molecular mechanism of general anesthesia. The results show that, besides its main preference for staying in the middle of the membrane, xenon also has a secondary preference for positions at the outer boundary of the hydrocarbon region, close to the crowded domain of the polar headgroups. This outer preference is a result of the interplay of the increasing attraction the xenon atoms experience and the decreasing free volume available for them upon going away from the middle of the membrane. It also turns out that any change that might be relevant in explaining the anaesthetic effect is related to xenon atoms staying in this outer preferred position. Since the model we employed does not account for the polarizability of the xenon atoms, and hence misses a part of the thermodynamic driving force of the preference for this outer position, all results should be considered as a lower bound estimate of the real effect. It is found that the xenon atoms induce a lateral swelling of the membrane by pushing the DPPC molecules farther away from each other, which, due to the insensitivity of the membrane thickness to the presence of xenon, naturally results in an increase of also the membrane volume. On the other hand, pressure increases the membrane thickness by increasing the order of the lipid tails, while it also causes a lateral shrinking of the bilayer. The net effect of these two opposite contributions is still a decrease of the membrane volume. In the light of these results, we propose to refine the 70 years old ‘critical volume hypothesis’ to a ‘critical surface area hypothesis’, claiming that general anesthesia occurs if the molar surface area of the membrane exceeds a critical value. Finally, the xenon-induced lateral swelling results in an increase of the empty space in the hydrocarbon region, allowing the DPPC molecules to move more freely, and hence increasing their lateral diffusion coefficient. All these changes are clearly reverted by pressure, and thus may well be relevant in respect of the molecular mechanism of general anesthesia.

Molecular Mechanism of General Anesthesia

Genel anestezik ajanlar, norotransmitterleri module ederek santral sinir sisteminde yaygin noronal degisime neden olmaktadir. Yeni yapilan molekuler arastirmalarda anestezik ajanlarin etki ettigi spesifik alanlar uzerinde durulmaktadir. Hipnoz, amnezi, sedasyon santral sinir sisteminde farkli reseptor, norotransmitter ve noronal yolaklar araciligiyla saglanir. 1980'lerin basinda yapilan calismalar sonrasinda, iyon kanallarina odaklanan protein temelli anestezi teorisi, lipid temelli anestezi teorisinin yerini almistir. Protein temelli teoriye gore, genel anestezik etkiden sorumlu iki tip reseptor mevcuttur; norotransmitter reseptorler ve iyon kanallari. “Background” iyon kanallari da anestezik etki icin yeni tanimlanmis hedef reseptorlerdir. Iki porlu (two-pore-domain) potasyum kanallarindan TWIK iliskili K+ kanali (TREK), TWIK iliskili arasidonik asitle aktive K+ kanali (TRAAK), TWIK iliskili asit duyarli K+ kanallari (TASK) tipteki kanallarin volatil ajanlarin dusuk konsantrasyonunda guclendigi ya da bloke oldugu bildirilmistir. Iki porlu potasyum kanallari, biyolojik membranlarda bulunan protein kompleksleridir. Iki porlu potasyum kanallari, potasyum iyonlari icin ozeldir ve potasyumun hucre membranindan gecmesine izin verirler. Iki porlu potasyum kanallari, noronal uyarilma, norotransmitter ve hormon salinimi yoluyla, membran potansiyelindeki degisikliklerden de sorumludurlar. Iki porlu potasyum kanallarin TASK-1 ve TREK-1 alt tipleri volatil anestezik ajanlarla (ornegin, izofluran, kloroform, dietil eter) uyarilir. Bu makalede santral sinir sisteminde bulunan ve genel anesteziklerin etkisinden sorumlu reseptorler ve bunlarin etki mekanizmalari gozden gecirilecektir.

Electron spin changes during general anesthesia in Drosophila.

We show that the general anesthetics xenon, sulfur hexafluoride, nitrous oxide, and chloroform cause rapid increases of different magnitude and time course in the electron spin content of Drosophila. With the exception of CHCl3, these changes are reversible. Anesthetic-resistant mutant strains of Drosophila exhibit a different pattern of spin responses to anesthetic. In two such mutants, the spin response to CHCl3 is absent. We propose that these spin changes are caused by perturbation of the electronic structure of proteins by general anesthetics. Using density functional theory, we show that general anesthetics perturb and extend the highest occupied molecular orbital of a nine-residue α-helix. The calculated perturbations are qualitatively in accord with the Meyer-Overton relationship and some of its exceptions. We conclude that there may be a connection between spin, electron currents in cells, and the functioning of the nervous system.

Atomistic study of lipid membranes containing chloroform: looking for a lipid-mediated mechanism of anesthesia.

The molecular mechanism of general anesthesia is still a controversial issue. Direct effect by linking of anesthetics to proteins and indirect action on the lipid membrane properties are the two hypotheses in conflict. Atomistic simulations of different lipid membranes subjected to the effect of small volatile organohalogen compounds are used to explore plausible lipid-mediated mechanisms. Simulations of homogeneous membranes reveal that electrostatic potential and lateral pressure transversal profiles are affected differently by chloroform (anesthetic) and carbon tetrachloride (non-anesthetic). Simulations of structured membranes that combine ordered and disordered regions show that chloroform molecules accumulate preferentially in highly disordered lipid domains, suggesting that the combination of both lateral and transversal partitioning of chloroform in the cell membrane could be responsible of its anesthetic action.

Novel Activation of Voltage-gated K+ Channels by Sevoflurane

Halogenated inhaled anesthetics modulate voltage-gated ion channels by unknown mechanisms. Biophysical analyses revealed novel activation of K(v) channels by the inhaled anesthetic sevoflurane. K(v) channel activation by sevoflurane results from the positive allosteric modulation of activation gating. The unique activation of K(v) channels by sevoflurane demonstrates novel anesthetic specificity and offers new insights into allosteric modulation of channel gating. Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05-1 mM) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (K(D) = 0.06 and 4 mM, respectively). Sevoflurane (<1 mM) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators.

Structural Studies of the Actions of Anesthetic Drugs on the γ-Aminobutyric Acid Type A Receptor

The γ-aminobutyric acid type A receptor is the major transmitter-gated inhibitory channel in the central nervous system. The receptor is a target for anesthetics, anticonvulsants, anxiolytics, and sedatives whose actions facilitate the flow of chloride ions through the channel and enhance the inhibitory tone in the brain. Both the kinetic and structural aspects of the actions of modulators of the γ-aminobutyric acid type A receptor are of great importance to understanding the molecular mechanisms of general anesthesia. In this review, the structural rearrangements that take place in the γ-aminobutyric acid type A receptor during channel activation and modulation are described, focusing on data obtained using voltage-clamp fluorometry. Voltage-clamp fluorometry entails the binding of an environmentally sensitive fluorophore molecule to a site of interest in the receptor, and measurement of changes in the fluorescence signal resulting from activation- or modulation-elicited structural changes. Detailed investigations can provide a map of structural changes that underlie or accompany the functional effects of modulators.

Visualization of the General Anesthetic Site in a K+ Channel

The structural basis of general anesthesia has remained hypothetical. Ion channels are plausible targets, as general anesthetics interact directly with neuronal ion channels and modulate their function. Investigating these interactions at the molecular level will further the understanding of general anesthesia and aid in the design of safer and more effective general anesthetics. The voltage-gated K+ channel Shaw2 is inhibited by relevant doses of n-alcohols and inhaled anesthetics at a discrete site. To map the residues that constitute this site in the activation gate of the channel, we conducted alanine-scanning mutagenesis in the S4-S5 linker and the S6 segment (post PVPV) of the Shaw2 protein and expressed the resulting mutants in Xenopus oocytes. Characterization of the 1-butanol and halothane dose-inhibition relations under voltage-clamp conditions and double mutant cycle analysis revealed significant energetic effects of single and double mutations on the inferred drug binding. Double mutants, such as Q320A/Y420A and A326V/Y419A (between the S4-S5 and S6), and Q320A/A326V (within the S4-S5 linker), exhibited the following coupling coefficients: 1.6, 4.3 and 2.8, respectively. Additional mutations suggested that these effects are not the result of altered gating. Exploiting these results, molecular dynamics simulations and docking calculations demonstrate the structural contributions of the S4-S5 linker and the distal S6 segment to a putative amphiphilic cavity that binds general anesthetics in the Shaw2 channel. Photolabeling of the Shaw2 protein is currently being explored as a method to test the hypothesis that this cavity is an anesthetic binding site. In conclusion, this work provides compelling evidence for a molecular mechanism of general anesthesia in which inhaled anesthetics modulate function through direct binding to a discrete cavity in the ion channel protein. Supported by NIH T04324 (AF), CNPg 141009/2009-8 (CA), NIH GM55876 (RGE), NIH AA010615 (MC).

Molecular mechanisms of general anesthesia

General anesthetics produce a widespread neurodepression in the central nervous system by enhancing inhibitory neurotransmission and reducing excitatory neurotransmission. However, the action mechanisms of general anesthetics are not completely understood. Moreover, the general anesthetic state comprises multiple components (amnesia, unconsciousness, analgesia, and immobility), each of which is mediated by different receptors and neuronal pathways. Recently, neurotransmitter- and voltage-gated ion channels have emerged as the most likely molecular targets for general anesthetics. The γ-aminobutyric acid type A (GABAA) receptors are leading candidates as a primary target of general anesthetics. This review summarizes current knowledge on how anesthetics modify GABAA receptor function.

Neuroscience of water molecules: a salute to professor Linus Carl Pauling

More than 35 years ago double Nobel laureate Linus Carl Pauling published a powerful model of the molecular mechanism of general anesthesia, generally referred to as the hydrate-microcrystal (aqueous-phase) theory. This hypothesis, based on the molecular behavior of water molecules, did not receive serious attention during Pauling's life time, when scientific tools for examining complex systems such as the brain were still in their infancy. The situation has since drastically changed, and, now, in the twenty first century, many scientific tools are available for examining different types of complex systems. The discovery of aquaporin-4, a subtype of water channel abundantly expressed in glial systems, further highlighted the concept that the dynamics of water molecules in the cerebral cortex play an important role in important physiological brain functions including consciousness and information processing.

Anesthetic Modulation of Protein Dynamics: Insight from an NMR Study

Mistic (membrane integrating sequence for translation of integral membrane protein constructs) comprises the four-alpha-helix bundle scaffold found in the transmembrane domains of the Cys-loop receptors that are plausible targets for general anesthetics. Nuclear magnetic resonance (NMR) studies of anesthetic halothane interaction with Mistic in dodecyl phosphocholine (DPC) micelles provide an experimental basis for understanding molecular mechanisms of general anesthesia. Halothane was found to interact directly with Mistic, mostly in the interfacial loop regions. Although the presence of halothane had little effect on Mistic structure, (15)N NMR relaxation dispersion measurements revealed that halothane affected Mistic's motion on the microsecond-millisecond time scale. Halothane shifted the equilibrium of chemical exchange in some residues and made the exchange faster or slower in comparison to the original state in the absence of halothane. The motion on the microsecond-millisecond time scale in several residues disappeared in response to the addition of halothane. Most of the residues experiencing halothane-induced dynamics changes also exhibited profound halothane-induced changes in chemical shift, suggesting that dynamics modification of these residues might result from their direct interaction with halothane molecules. Allosteric modulation by halothane also contributed to dynamics changes, as reflected in residues I52 and Y82 where halothane introduction brought about dynamics changes but not chemical shift changes. The study suggests that inhaled general anesthetics could act on proteins via altering protein motion on the microsecond-millisecond time scale, especially motion in the flexible loops that link different alpha helices. The validation of anesthetic effect on protein dynamics that are potentially correlated with protein functions is a critical step in unraveling the mechanisms of anesthetic action on proteins.

Four- α-Helix Bundle with Designed Anesthetic Binding Pockets. Part I: Structural and Dynamical Analyses

The four- α-helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four- α-helix bundle, (A α 2-L1M/L38M) 2, with designed specific binding pockets for volatile anesthetics. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. The two helices from the N-terminus to the linker (helices 1 and 1′) are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2′) are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The site of the L38M mutation, which was previously shown to increase the halothane binding affinity by ∼3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R 2) rate. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia.

The molecular mechanisms of general anaesthesia: dissecting the GABAA receptor

The molecular mechanisms of general anaesthesia: dissecting the GABAA receptor

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.

Optimization of Structures and LJ Parameters of 1-Chloro-1,2,2-trifluorocyclobutane and 1,2-Dichlorohexafluorocyclobutane

The structures and the Lennard-Jones (LJ) parameters of anesthetic 1-chloro-1,2,2-trifluorocyclobutane (F3) and nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6) were optimized by using ab initio calculations in conjunction with liquid and gas phase molecular dynamics simulations. Geometry optimization of various isomers of F3 and F6 was carried out with MP2/6-311+G(2d,p) that reproduced the experimental pucker angles of the precursors perfluorocyclobutane and cyclobutane more accurately than with B3LYP/6-311+G(2d,p). Frequency calculations were performed to ensure that the optimized structures were at minimums of the potential energy surfaces. The partial atomic charges of F3 and F6 from the Merz−Singh−Kollman MP2/6-311+G(2d,p) calculations and the LJ parameters optimized previously for other halogenated compounds were used to start molecular dynamics simulations. The LJ parameters were then optimized through iterative adjustments to regenerate the heat of vaporization and the densities of F3 and F6 until the differences between the calculated values and the experimental values or empirical predicted values were less than 2%. With the optimized structures, the partial atomic charges, and the LJ parameters, F3 and F6 are readily usable as an anesthetic−nonimmobilizer pair in molecular dynamics simulations aimed at understanding the molecular mechanisms of general anesthesia.

Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: the implication of molecular mechanisms of general anesthesia.

Interactions of volatile anesthetics with the central nervous system are characterized by low yet specific binding affinities. Although neurotransmitter-gated ion channels are considered the primary anesthetic targets, the mechanism of action at the molecular level remains elusive. We consider here the theoretical implications of channel dynamics on anesthetic action in a simplified membrane-channel system. Large-scale 2.2-ns all-atom molecular dynamics simulations were performed to study the effects of halothane, a clinical anesthetic, on a gramicidin A (gA) channel in a fully hydrated dimyristoyl phosphatidylcholine membrane. In agreement with experimental results, anesthetics preferentially target the anchoring residues at the channel-lipid-water interface. Although the anesthetic effect on channel structure is minimal, the presence of halothane profoundly affects channel dynamics. For 2.2-ns simulation, the rms fluctuation of gA backbone in the lipid core increases from approximately equal 1 A in the absence of anesthetics to approximately equal 1.5 A in the presence of halothane. Autocorrelation analysis reveals that halothane (i) has no effect on the subpicosecond librational motion, (ii) prolongs the backbone autocorrelation time in the 10- to 100-ps time scale, and (iii) significantly decreases the asymptotic values of generalized order parameter and correlation time of nanosecond motions for the inner but not the outer residues. The simulation results discount the viewpoint of a structure-function paradigm that overrates the importance of structural fitting between general anesthetics and yet-unidentified hydrophobic protein pockets. Instead, the results underscore the global, as opposed to local, effects of anesthetics on protein dynamics as the underlying mechanisms for the action of general anesthetics and possibly of other low-affinity drugs.

Effects of Volatile Anesthetic on Channel Structure of Gramicidin A

Volatile anesthetic agent, 1-chloro-1,2,2-trifluorocyclobutane (F3), was found to alter gramicidin A channel function by enhancing Na + transport (Tang et al. 1999. Biophys. J. 77:739–746). Whether this functional change is associated with structural alternation is evaluated by circular dichroism and nuclear magnetic resonance spectroscopy. The circular dichroism and nuclear magnetic resonance results indicate that at low millimolar concentrations, 1-chloro-1,2,2-trifluorocyclobutane causes minimal changes in gramicidin A channel structure in sodium dodecyl sulfate micelles. All hydrogen bonds between channel backbones are well maintained in the presence of 1-chloro-1,2,2-trifluorocyclobutane, and the channel structure is stable. The finding supports the notion that low affinity drugs such as volatile anesthetics and alcohols can cause significant changes in protein function without necessarily producing associated changes in protein structure. To understand the molecular mechanism of general anesthesia, it is important to recognize that in addition to structural changes, other protein properties, including dynamic characteristics of channel motions, may also be of functional significance.

Molecular genetical analysis of Drosophila ether sensitive mutants

1. For a genetical approach to the molecular mechanisms of general anesthesia, we isolated 14 Drosophila transposon tagging mutants which showed altered sensitivities to diethylether anesthesia. Two mutated genes were identified and analyzed for their structures and functions. 2. One sensitive mutant, para hd 838, has a transposon, P-element, insertion in the 2nd intron of para locus (14C) which encodes an α subunit of a sodium channel. 3. Another sensitive mutant, eth as311, has a P-element in the 5′-untranslated region of Drosophila calreticulin gene (86BC). Calreticulin is known as a multifunctional calcium-binding protein in the endoplasmic reticulum of non-muscle cells. 4. We obtained their revertants to show that the P-element insertion in each gene induced the hypersensitivity to ether anesthesia. We describe here that the sodium channel and calreticulin might be targets for the ether anesthesia.

Keith W. Miller

Keith W. Miller

Steady-state catecholamine distribution in chromaffin granule preparations: a test of the pump-leak hypothesis of general anesthesia

The molecular mechanism of general anesthesia is not understood. Possible modes of action include binding at a protein site, such as a receptor or channel, or physical effects on membrane lipid properties. The pump-leak hypothesis suggests that anesthetics perturb the bilayer of synaptic vesicles, thereby increasing ionic permeability. This results in decay of proton gradients required for transport and accumulation of neurotransmitters. The subsequent loss of neurotransmitters from synaptic vesicles reduces the efficiency of synaptic transmission and results in the anesthetized state. We have determined the effects of general anesthetics on certain parameters of enzyme activity and membrane permeability relevant to the pump-leak hypothesis. We used chromaffin granules as a convenient model system and focused on clinically relevant anesthetic concentrations (ED50), quantitative measurements of permeability changes, and the kinetics of gradient decay. General anesthetics at ED50 have little or no effect on the proton-transport ATPase activity, but do cause modest increments in proton permeability that change the catecholamine distribution in actively pumping chromaffin granule preparations. We found that pH gradients do not collapse entirely under these conditions and that only a fraction of total catecholamine is lost from the chromaffin granules. When total collapse is induced by other means, efflux of catecholamines occurs with a half-time near 30 min. These results suggest that if the pump-leak hypothesis is valid, then very small losses of catecholamines must be sufficient to induce anesthesia. We conclude that the weight of evidence favors other mechanisms, notably direct binding of anesthetics to sensitive proteins.