Molecular modelling of specific and non-specific anaesthetic interactions

J. R. Trudell* and E. Bertaccini

Department of Anaesthesia and Beckman Program for Molecular and Genetic Medicine, Stanford University, Stanford, CA 94305-5117, USA*Corresponding author

Abstract

Br J Anaesth 2002; 89: 32–40

Keywords: ions, ion channels; theories of anaesthetic action

The appropriate point of focus for the molecular modelling of anaesthetic sites of action has moved through an interesting circle during the last century. The early correlation of anaesthetic potency with solubility in moderately polar solvents46 55 suggested theories of anaesthetic action in hydrophobic regions based on relatively non-specific colligative properties of solvents. The picture of biological membranes with proteins imbedded in and passing through phospholipid bilayers that emerged in the late 1960s71 suggested phospholipid bilayers44 48 80 and the interface of phospholipid bilayers with intrinsic membrane proteins28 78 as more specific targets of anaesthetic action. However, at nearly the same time, the first x-ray structure of the inhalation anaesthetic, halothane, bound to a specific site in adenylate kinase appeared.64 This example of specific binding was followed by the crystal structures of xenon and chloroform bound to specific sites in myoglobin.67 68 76 A turning point in the focus of anaesthetic action was the demonstration that anaesthetic inhibition of a lipid-free enzyme, luciferase, correlated with in vivo anaesthetic potency.19 The focus of molecular modelling was further shifted by recent crystal structures of anaesthetics bound to specific sites in luciferase,20 cholesterol oxidase9 and albumin.10 Most recently, molecular biology has revealed that the amino acid residues that confer sensitivity to anaesthetics in ligand-gated ion channels are located in the transmembrane domain.47 101 Therefore, we once again must consider sites of anaesthetic action in regions of nerve membranes where there is intimate contact and interaction between proteins and lipids.

However, a different expectation for the dynamics of anaesthetic molecules in their binding sites is an important aspect of this change in focus from non-specific action in lipids to stereospecific action at protein sites. When we first used F-19 NMR to measure the rate of exchange of a halothane molecule between the aqueous region and the phospholipid bilayer, we were amazed that the rate of exchange was faster than 1 m s–1.81 Furthermore, with the use of spin-labelled phospholipids, we were able to show that the penetration of halothane molecules extended into the centre of the bilayer. Our initial estimates of the rate of exchange were revealed by much more sophisticated NMR techniques to be in the microsecond time domain.75 100 In contrast, the techniques of x-ray crystallography require minutes to hours of exposure of a sample to obtain an image. Therefore, in order for an anaesthetic molecule to be imaged as discrete atoms in the x-ray structure, the atoms must remain essentially motionless during the exposure at a resolution of approximately 2 Å. This implies that, even if the anaesthetic molecules are in rapid equilibrium with the surrounding solvent, they must return to the same atomic co-ordinates when they re-enter the binding site.

This relatively static binding has important consequences for molecular modelling: if anaesthetic molecules are to remain at specific co-ordinates, the large entropic penalties of this restriction in position must be overcome by corresponding enthalpic contributions25 83 that probably exceed those that can derive solely from dispersion forces of a molecule in an isotropic solvent.2 That is, there must be explicit electrostatic bonds, hydrogen bonds or charge-induced dipoles that contribute to the anaesthetic–binding site interaction.83 This point was reviewed by Katz and Simon in their analysis of the anaesthetic potency of the noble gases.35

A new point of motivation for the molecular modelling of anaesthetic binding is that the goal of an agent that rapidly reverses anaesthetic or alcohol intoxication may be a reality. In a recent publication, Beckstead and co-workers5 have carefully selected a mutant glycine receptor that was insensitive to the enhancing effects of ethanol but remained sensitive to enflurane, toluene and chloroform. Although ethanol (25–200 mM) had no effect on its own in this receptor, it was able to inhibit reversibly the enhancing effect of enflurane, toluene and chloroform in a concentration-dependent manner. The implications of this experiment are that (i) it may be possible for molecular modelling of a binding site to define an agent that would reverse anaesthetic or alcohol intoxication; and (ii) it appears that dissimilar anaesthetics and alcohols can occupy similar or adjacent sites in a specific receptor.

These results suggest that molecular modelling could be applied to specific sites and could define those differences in binding sites that allow some molecules to bind without effect and yet prevent other, normally efficacious, molecules from having their usual effect. That is, the steric and electrostatic properties of anaesthetic binding sites, as derived from molecular modelling studies, may be sufficiently specific to allow design of drugs that can rapidly reverse the effects of anaesthetics and alcohol. This article will discuss the most recent progress in these areas, which includes the modelling of anaesthetics with lipid bilayers, of synthesized four-helical protein bundles, of gramicidin A embedded within a phospholipid bilayer, and of the transmembrane domain of ligand-gated ion channels.

Molecular modelling of anaesthetic effects on phospholipid bilayers

We should not be too quick to focus on specific protein binding sites because there is additional evidence for the influence of lipid–protein interactions in anaesthetic interactions. As reviewed below, site-directed mutations in ligand-gated ion channels suggest that a primary point of action for anaesthetics is in the transmembrane domain of these channels.47 101 That is, the anaesthetic binding sites are directly adjacent to the phospholipid bilayer and it is possible that fatty acid chains form part of the boundary of the sites. In addition, there is much evidence for a strong effect of the properties of lipid bilayers on the function of intrinsic membrane proteins. One example is that bovine rhodopsin reconstituted in phospholipid vesicles is only functional when the fatty acid chain length is C-14 or greater.3 Another is that the nicotinic acetylcholine receptor requires cholesterol for correct function60 and that this cholesterol is intimately associated with the receptor.15 34 The role of membrane lipids in controlling conformational changes in intrinsic membrane proteins13 78 will be especially important in conformationally mobile proteins such as ion channels.27

Recent molecular dynamics studies of Sharf and co-workers31 84 are in accordance with earlier F-19 NMR studies of anaesthetic interactions with lipid bilayers.75 79 104 They show that anaesthetic molecules are distributed throughout the phospholipid bilayer but are preferentially localized near the phospholipid head groups. Their molecular dynamic simulations studied the distribution of anaesthetic molecules within a complete phospholipid bilayer composed of hundreds of explicit phospholipid molecules that were hydrated at both interfaces with thousands of explicit water molecules. This hydrated bilayer was, in turn, placed in a periodic box of water molecules which conferred the property that if one water molecule leaves the box heading in, for example, the –x direction, it is replaced by a molecule entering from the +x direction. This periodic box has the important function that it allows the investigator to control the absolute pressure in the system and to maintain a specified temperature (thermal kinetic energy) and initial number of anaesthetic, phospholipid and water molecules.31 84

Tu and colleagues84 performed constant temperature and pressure molecular dynamic calculations on the liquid crystal (L-{alpha}) phase of dipalmitoylphosphatidylcholine with a mole fraction of 6.5% halothane (2–3 MAC). Subtle structural changes in the lipid bilayer occurred in the presence of the anaesthetic, compared with the pure lipid bilayer, such that a small lateral expansion accompanied by a modest contraction in the bilayer thickness was found. However, the overall increase in the system volume was found to be comparable to the molecular volume of the added anaesthetic molecules, in agreement with previous studies.77 No significant changes in the hydrocarbon chain conformations were apparent. The observed structural changes were in fair agreement with NMR data corresponding to low anaesthetic concentrations. It was found that halothane exhibited no specific binding to the lipid head-groups or to the acyl chains. No evidence was obtained for preferential orientation of halothane molecules with respect to the lipid/water interface.

Binding of anaesthetic molecules in four-helical bundles

Experimental support for the hypothesis that small molecules can bind in cavities formed between {alpha}-helices was recently provided by Johansson and co-workers.30 32 33 They designed a binding cavity in the hydrophobic core of a four-{alpha}-helix bundle (Fig. 1). Replacing six core leucine residues with alanine provided a cavity of approximately 171 Å3 (halothane has a calculated van der Waals volume of 123 Å3), which increased halothane binding affinity 4.4-fold. Structural changes in the bundle caused by binding were measured with circular dichroism, ESR, and fluorescence spectroscopy. A molecular dynamics simulation of this bundle in a phospholipid bilayer demonstrated improved binding sites for halothane.16 Nanosecond length molecular dynamic trajectories were generated for each system at room temperature (298 K). The structural and dynamic effects of the inclusion of halothane were compared, illustrating that the structures are stable over the course of the simulation, that the four-{alpha}-helical bundles have suitable pockets that can accommodate halothane, that halothane remains in the designed hydrophobic cavity in close proximity to the tryptophan residues with a preferred orientation, and that the dimensions of the peptide are perturbed by the inclusion of an anaesthetic molecule



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Fig 1 Side-view of two di-{alpha}-helical peptides that dimerize to form a four-{alpha}-helix bundle. The {alpha}-helical backbones are shown, along with space-filling renditions of methionine 38 (at ends), tryptophan 15 (in centre) and a halothane molecule in darker grey.

 
These authors then used their model to probe structural features of binding cavities that may determine anaesthetic potency. They showed that substitution of methionine residues in the four-{alpha}-helical bundles further increased binding affinity.31 33 The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein.33 The resulting helical bundle variant had an improved affinity (K(d)=0.20±0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d)=0.69±0.06 mM). Photoaffinity labelling with [14C]-halothane revealed preferential labelling of the tryptophan residue (W15) in both peptides, supporting the view that fluorescence quenching by bound anaesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-{alpha}-helical bundle proteins stabilized the native folded conformations. Molecular dynamic simulations of the bundles illustrated the existence of the hydrophobic core, containing both W15 residues.16 33 These results suggest that, in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anaesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site, or allosteric optimization of the dimensions of the binding pocket. Finally, the results suggest that preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anaesthetic action.

Molecular modelling of the effects of halothane on gramicidin A embedded in phospholipid bilayers

Tang and Xu have used both Xe-12999 and F-1974 high resolution NMR to define the environments and positions of anaesthetic molecules in model systems of gramicidin reconstituted in phospholipid bilayers. They recently used the latter technique to show that a pair of structurally similar compounds, the volatile anaesthetic [1-chloro-1,2,2-trifluorocyclobutane (F3)] and a non-immobilizer (non-anaesthetic) [1,2-dichlorohexafluorocyclobutane (F6)], interact differently with the transmembrane surface of gramicidin.73

These authors subsequently used molecular dynamic simulations to study the dynamic properties of anaesthetic molecules in the same system.74 107 In these simulations, they used gramicidin A as a model for a transmembrane ion channel. They investigated whether interfacial lipids (that is, those at the gramicidin channel–membrane interface) play a significant role in mediating anaesthetic effects on the ion channel. Large-scale, all-atom molecular dynamic simulations of a gramicidin channel in a fully hydrated dimyristoylphosphatidylcholine (DMPC) bilayer were performed in the presence and absence of halothane using the NAMD2 molecular dynamics programme (University of Illinois at Urbana-Champaign, USA) on the T3E supercomputer at the Pittsburgh Supercomputing Centre. A gramicidin channel (an end-to-end dimer that spans the membrane) was placed in a DMPC–water system and molecular dynamics was performed until the system had reached thermal equilibrium and constant pressure. Then 10 halothane molecules were placed at predetermined locations based on their NMR results.74 After additional equilibration and energy minimization, parallel molecular dynamic simulations with and without halothane were carried out for 2.2 ns each. The simulations revealed intimate details of how halothane might affect a gramicidin channel and the lipid membrane. Figure 2 shows the initial distribution of halothane molecules in the dynamics simulation.



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Fig 2 A snapshot of the simulation system: a gramicidin channel in fully hydrated DMPC membrane with 10 halothane molecules at their initial positions. For clarity, DMPC lipid chains are not shown. The phosphorus atoms in the lipid head groups are marked with black spheres.

 
The transmembrane domain of ligand-gated ion channels as a target for molecular modelling

Although the following sections will concentrate on the effect of anaesthetics on ligand-gated ion channels, other reviews in this volume make it clear that these channels provide one, but not the only, site for molecular modelling. The cellular systems that are affected by anaesthetics and alcohol are almost too numerous to mention.101 Although there have been many attempts to find one ion channel, membrane site or enzyme that is ‘most sensitive’, it appears that it is more reasonable to consider intoxication or anaesthesia as a sum of many contributions. The diversity of ion channels affected by anaesthetics includes potassium channels,38 91 102 105 calcium channels (both voltage and ligand-sensitive),90 94 glutamate receptors (both NMDA56 and kainate49), the novel P2X(4) receptor,98 nicotinic acetylcholine receptors (nAChR),93 106 5HT3 channels,17 GABAA channels,69 85 88 92 97 103 and glycine (GlyR{alpha}1) channels.43 47 An important aspect of anaesthetic action involves modulation of protein function by kinases and phosphatases, for example the effect of local anaesthetics on phosphorylation by protein kinase C.81 82 More recent work has shown specific effects of anaesthetics and alcohol on phosphorylation of important cellular targets by protein kinase C.11 62 63 72 90 92 The role of ethanol in inhibition of autophosphorylation of IGF-I and insulin receptors by tyrosine kinases has also been investigated.70

Is the transmembrane domain of ligand-gated ion channels composed of five subunits, each of which is a tetrameric bundle of {alpha}-helices?
In general, transmembrane domains of proteins have been experimentally shown to be either {alpha}-helical bundles or ß-barrels.89 A standard hypothesis is that the common motif of the ligand-gated ion channel (LGIC) superfamily is a pentamer of subunits, with each subunit consisting of four anti-parallel {alpha}-helices.68 40 101 The general motif of an ion channel composed of five subunits arranged around a central pore is strongly supported by a series of papers by Unwin and co-workers.50 86 The suggestion that the other three transmembrane segments in each subunit are also {alpha}-helices is much more controversial.1 21 36 37 54 86 95

Many techniques have been used to predict the secondary structure of the transmembrane regions of LGICs.4 6 21 22 36 39 54 101 Because a consensus prediction of secondary structure was essential before building a molecular model of putative binding sites in LGICs, Bertaccini and Trudell6 8 made a substantial effort to make such a prediction using modern algorithms specifically designed to predict {alpha}-helices in membrane (not globular) proteins. The amino acid sequences of six LGICs were obtained from the Entrez protein database at the US National Library of Medicine: (i) Torpedo nicotinic acetylcholine receptor {alpha} (Torpedo AChR{alpha}1), (ii) Human neuronal nicotinic acetylcholine receptor {alpha} 4 (AChR{alpha}4), (iii) Human neuronal nicotinic acetylcholine receptor {alpha} 7 (AChR{alpha}7), (iv) Human GABA {alpha} 1 receptor (GABAR{alpha}1), (v) Human glycine {alpha} 1 receptor (GlyR{alpha}1) and (vi) Human 5-hydroxytryptamine receptor (5HT3). Each protein sequence was submitted in total to each of 10 transmembrane protein topology prediction algorithms. The clear consensus of the 60 computations was that the transmembrane domain of the AChR superfamily of ligand-gated ion channels was composed of subunits containing four-{alpha}-helical bundles.

There is now considerable experimental support for this computational result. Recently, Methot and colleagues45 conducted an elegant Fourier transform infrared (FTIR) analysis of the Torpedo AChR. They reconstituted Torpedo AChR in a liposomal preparation and demonstrated a mixed content of {alpha}-helix and ß-sheet in the whole receptor. They then exposed the receptor to proteolytic cleavage both external and internal to the liposome to leave only the transmembrane component of the protein embedded in the liposomal membrane. Subsequent FTIR analysis clearly demonstrated a preponderance of {alpha}-helical character. In fact, the only hint of ß-sheet character found in this portion was similar to that noted in other proteins that have been shown to be entirely {alpha}-helical in structure by x-ray crystallography.

A molecular model of a ligand-gated ion channel of the AChR superfamily
Based on the result of secondary structure prediction in the AChR superfamily,8 a model of the transmembrane domain of a homopentameric GABA{alpha}R {alpha}-1 ion channel was built.7 101 Each subunit was formed by threading the amino acid sequence of GABA{alpha}R {alpha}-1 onto the crystal structure of a four-{alpha}-helical bundle found in cytochrome oxidase. Assembly of five of these subunits to form a homopentameric ion channel was based on the threading of TM2 within each subunit onto the crystal structure of a bacterial mechanosensitive ion channel (1msl)14. This ion channel is believed to be a primordial progenitor for the majority of ion channels in higher organisms. This homopentameric ion channel contains five {alpha}-helices arranged around a central pore with a right-hand supertwist and a funnel shape that is narrowest at the intracellular face. The structure supports the {alpha}-helical nature of TM2 in LGICs and is consistent with our predictions as well as those based on electron density in the cryoelectron micrographs of acetylcholine receptors by Unwin and colleagues.50 86 87 Although early studies predicted a ‘kink’ at the highly conserved leucine residue lining the ion channel,86 87 this kink was not observed in a recent NMR study.53 Moreover, application of simulated annealing via restrained molecular dynamics (SA/MD) to a model of the AChR ion channel showed that the kink may be achieved by cumulative small distortions of the backbone from canonical {alpha}-helical geometry, rather than a marked loss of {alpha}-helical geometry in the vicinity of the conserved leucine.65

Although the resulting model containing 20 transmembrane {alpha}-helices satisfied much of the available experimental data,1 7 36 45 47 95 96 it was desirable to test how well the bacterial mechanosensitive ion channel would serve as a template for the AChR superfamily of ion channels. A means to test the quaternary structure of the model was provided by the recent publication of the crystal structure of the acetylcholine binding protein.12 This protein was shown to have high homology to portions within members of the AChR superfamily, including human and Torpedo AChRs, GABAAR and the glycine receptor. However, it was possible to crystallize this protein because it contains only the extracellular ligand-binding domain of the AChR. Since only the transmembrane domain had been modelled, it followed that, if the structure of our model was approximately correct, it should be possible to place the structure of the acetylcholine binding protein onto our model of the transmembrane domain and observe how well they fit together. This test is shown in Figure 3. One can see that the supertwist of the five pore-lining {alpha}-helices (TM2 in AChR) and the outward flare of those helices to form a funnel-shaped pore narrowest at the intracellular side is well matched by the structure of the ligand-binding domain in the crystal structure. This point is particularly striking in the top view (from the extracellular side) where one can observe the vestibule formed by the acetylcholine-binding protein and the smooth transition into the lumen of the ion channel in our model of the transmembrane domain.



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Fig 3 (A) The crystal structure of the acetylcholine-binding protein (top) and a model of the transmembrane domain of the GABA alpha 1 receptor (bottom) viewed from the extracellular side of the membrane. (B) The two structures are juxtaposed by rotation of +90 degrees and –90 degrees, respectively. The test of the model at the bottom was to try to move the crystal structure of the ligand-binding domain onto the transmembrane domain and observe how well the model fits the crystal structure. (C) The crystal structure was moved vertically until in van der Waals contact with the model as shown by the horizontal lines that mark the lipid bilayer region of the transmembrane domain. The image in (C) is rendered in space-filling (van der Waals) volume. A site of anaesthetic insertion is labelled ‘interface’. It can be seen that the fit of the model, which was based on the crystal structure of the bacterial mechanosensitive channel (1msl), with the crystal structure of the acetylcholine binding protein is very good.

 
Two new features (at least, new to the authors) were highlighted by this model. First, while juxtaposing the two structures, it became clear that the ‘cis-loop’ at the bottom face of the acetylcholine-binding protein (a loop fixed by a disulfide bond between two cysteines that are completely conserved throughout the AChR superfamily) is likely to interact with the loop that connects transmembrane {alpha}-helices 2 and 3 in the model of the transmembrane domain of LGICs, the ‘2–3 loop’.

Second, the composite model suggests that we may have to modify our view of how anaesthetic molecules (or meth anethiosulfonate reagents)1 95 could enter a subunit. When one views a model of the transmembrane domain of LGICs in isolation, it is easy to assume that these small molecules move directly from the extracellular fluid into a putative cavity formed between the {alpha}-helices or ß-strands that may make up a subunit. However, with the ligand-binding domain in place (Figure 3C), it is clear that this straightforward entry path is not possible. Therefore, one must think in terms of pathways that include either (1) diffusion down the water-filled lumen of the ion channel, (2) dissolution in the phospholipid bilayer followed by transfer through the lipid–protein interface of the ion channel or (3) transfer to an annular ring formed by the four-component interface of the ligand-binding and transmembrane domains of the protein, the phospholipid bilayer and the interfacial water layer. Interestingly, it is at the third possibility where Tang and Xu found localized halothane molecules in their studies described above.74 107

Modelling studies lead to a global model of how anaesthetic binding to a non-competitive site in a receptor could affect function of an ion channel

The large Kd of alcohol and inhalational anaesthetics (approximately 100 mM and 1 mM, respectively) require that their binding energies to sites of action are small. As a result, we should not expect that the binding event could cause an ‘induced fit’ in a protein site or even provide substantial reorganization of an internal cavity. Rather, it is likely that these molecules bind adventitiously to pre-existing cavities or sites.43 47 51 52 These sites may be essentially identical to those engineered into four-helical bundles by Johansson and co-workers.32 33 The binding of small molecules could affect receptor function if there is equilibrium between the conformations of resting channels and those in the open, desensitized state, with binding of a small ligand changing that equilibrium. One could further hypothesize that the ligand binds to most or all conformations of the receptor, but that the shapes or volumes of the binding sites differ between conformations. In that case, a given ligand could fit into a site in one conformation better than another and provide more stabilization to that conformation. This suggestion leads to the possibility that, on a molecular level, a site-directed mutation could change which conformation is most stabilized by a particular ligand. In fact, there are now several examples in which single mutations change the relative potency of alcohols and inhalational anaesthetics.23 24 29 59 66 Preliminary models of anaesthetic and alcohol binding sites include internal cavities in proteins.18 58 61 Often these sites are between transmembrane {alpha}-helices. Such binding sites are similar to the ‘domain interface model’ described by Catterall and colleagues for ligand binding to L-type calcium channels.57 The hydrophobic cavity created in T4 lysozyme by a leucine-to-alanine mutation at position 99 (L99A)51 52 is a good initial model for an anaesthetic and alcohol binding site for the following reasons: mutations in ligand-gated ion channels are relatively conservative; they involve mainly neutral amino acid residues; and the mutations may create or expand small hydrophobic cavities much like those created in T4 lysozyme. A particular advantage of using the cavity in T4 lysozyme as a reference is that binding of small molecules, and the resulting stabilization of protein structure, has been studied by NMR, thermal denaturation, microcalorimetry, X-ray diffraction,58 fluorescence41 52 and molecular dynamic simulations26 42.

Summary

There has been rapid progress in molecular modelling in recent years. The convergence of improved software for molecular mechanics and dynamics, techniques for chimeric substitution and site-directed mutations, and the first x-ray structures of transmembrane ion channels have made it possible to build and test models of anaesthetic binding sites. These models have served as guides for site-directed mutagenesis and as starting points for understanding the molecular dynamics of anaesthetic–site interactions. Ligand-gated ion channels are targets for inhaled anaesthetics and alcohols in the central nervous system.102 The inhibitory strychnine-sensitive glycine and {gamma}-aminobutyric acid type A receptors are positively modulated by anaesthetics and alcohols; site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anaesthetics and alcohols in these receptors. Key questions are whether these amino acid mutations form part of alcohol- or anaesthetic-binding sites or if they alter protein stability in a way that allows anaesthetic molecules to act remotely by non-specific mechanisms. It is likely that molecular modelling will play a major role in answering these questions.

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