Characterization of the binding of the local anesthetics procaine and tetracaine to model membranes of phosphatidylethanolamine: a deuterium nuclear magnetic resonance study

The interaction of the local anesthetics tetracaine and procaine with multilamellar dispersions of phosphatidylethanolamine has been investigated by using 2H NMR of specifically deuterated anesthetics. Tetracaine was found to partition more strongly than procaine into the lipid. The 2H NMR spectra showed a quadrupole doublet and a narrow line, with the former corresponding to membrane-bound anesthetic and the latter to anesthetic free in solution. The integrated areas of the narrow line and of the doublet correspond to the concentrations of free and bound anesthetic predicted from the Kp values. There is no strong pH dependence for the quadrupole splittings of tetracaine, suggesting a similar depth of penetration into the lipid bilayer over the entire pH range. The data are consistent with a model in which tetracaine acts as a wedge to stabilize the phosphatidylethanolamine bilayer against transition to a hexagonal structure. Procaine is proposed to sit higher in the phosphatidylethanolamine bilayer than does tetracaine. The T1 values were generally shorter in the membrane than in solution, suggesting slower motions, particularly for the aromatic ring of tetracaine.     

Interaction of fluorinated ether anesthetics with artificial membranes.

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

Interaction of fluorinated ether anesthetics with artificial membranes

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T₁) measurements are consistent with substantial immobilization of the lipid-bound anesthetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

Lipid protein interactions in mitochondria. VIII. Effect of general anesthetics on the mobility of spin labels in lipid vesticles and mitochondrial membranes

We have studied the effect of general anesthetics on the mobility of two stearic acid spin labels (5-doxyl stearic acid and 16-doxyl stearic acid) in bovine heart mitochondria and in phospholipid vesicles made from either mitochondrial lipids or commercial soybean phospholipids. The general anesthetics used include nonpolar compounds (alcohols, halothane, pentrane, diethyl ether, chloroform) and the amphipathic compound, ketamine. All anesthetics tested increase the mobility of the spin labels in phospholipid vesicles to a limited extent up to a concentration where the ESR spectra become those of free spin labels. On the other hand, anesthetics have a pronounced effect on mitochondrial membranes at concentrations as low as those known to produce general anesthesia; the effect is lower near the bilayer surface (5-doxyl stearic acid) and very strong in the bilayer core (16-doxyl stearic acid). The effects of anesthetics are mimicked by the detergent, Triton X-100. We suggest that the discrepancy between the action of anesthetics in mobilizing the spin labels in lipid vesicles and in membranes results from labilization of lipid protein interactions.     

Laser flash photolysis as a probe of redox protein-membrane interactions: effect of binding of spinach plastocyanin and horse cytochrome c to lipid bilayer vesicles on the kinetics of reduction by flavin semiquinone

Spinach plastocyanin binds to both electrically neutral and positively charged lipid bilayer vesicles, whereas cytochrome c only binds electrostatically to negatively charged vesicles. Laser flash photolysis using lumiflavin semiquinone as a reductant demonstrates that the reactivity of plastocyanin is increased as much as 6-fold when it is membrane bound whereas the rate constant for cytochrome c reduction is decreased by approximately a factor of 3. Membrane-bound plastocyanin reduction occurs via a two-step mechanism, probably involving prior association of lumiflavin semiquinone with the bilayer. In contrast, cytochrome c reduction in the membrane-bound state follows simple second-order kinetics, implying that the redox site in the bound state is still accessible to lumiflavin semiquinone in solution, although the rate constant is decreased by approximately 3-fold. These results are interpreted as indicating that the bilayer-protein interaction with plastocyanin leads to a steric blockage of the electron-transfer site from the aqueous phase. Little or no hindrance of the redox site occurs with cytochrome c, suggesting a high degree of mobility of this protein on the bilayer surface. Although the increase in plastocyanin reactivity upon binding to the bilayer is quite interesting, its cause remains unclear and requires further study. The results illustrate the utility of laser flash photolysis as a probe of membrane-protein interactions.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

The Proapoptotic Protein tBid Forms Both Superficially Bound and Membrane-Inserted Oligomers

Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine

Anesthetics bound to model membranes were observed directly by means of deuterium nuclear magnetic resonance (NMR). The specifically deuterated local anesthetics procaine and tetracaine were synthesized, and their partition coefficients (water:phosphatidylcholine) and pKa values determined. The interaction of these anesthetics with lamellar dispersions of egg phosphatidylcholine was studied by 2H nuclear magnetic resonance and by electron spin resonance (ESR) of a spin-labelled phospholipid at low (5.5) and high (9.5) pH. The ESR experiments suggest that tetracaine intercalates in the membrane and that it equilibrates between water and the phospholipid bilayers of the multilamellar system. The NMR results are consistent with a model where the anesthetic is (1) free in water, (2) weakly bound, and (3) strongly bound to the membrane. A fast exchange exists between the two first sites, but exchange is slow with the third site. Binding of type 3 is observed only at high pH for procaine, whereas it is found both at low and high pH for tetracaine. Calculations of the partition coefficients for the charged and uncharged forms of tetracaine indicate that both sites, 2 and 3, are occupied by the charged form at low pH and by the uncharged form at high pH. The partition coefficient for the weakly bound species was estimated from an analysis of the dependence of line width on the lipid to water ratio. The NMR data suggest that the binding sites for the strongly bound charged and uncharged species are different, the former probably being closer to the membrane-water interface. Estimates of molecular order parameters for the strongly bound species indicate that it is located with its long molecular axis approximately parallel to the director for ordering of the fatty acyl chains. A small increase in lipid ordering by tetracaine is observed at low pH, as evidenced by 2H NMR of the deuterated N-methyl groups of phosphatidylcholine; the reverse occurs at high pH.     

Local anesthetics can interact electrostatically with membrane proteins

The fluorescent probe 1-anilinonaphthalene 8-sulfonate was used to examine the binding of spin-labeled local anesthetics to lipid model systems, to the membranes of human red blood cells, and rabbit sarcoplasmic reticulum. 1-Anilinonaphthalene 8-sulfonate exhibits two distinct fluorescent lifetimes when bound to these biological membranes. The shorter lifetime represents the probe associated with the purely lipid region while the longer lifetime is associated with the protein region. The spin-labeled local anesthetic quenches the fluorescence of both of these components as indicated by the decrease in the lifetimes. Since nitroxide free radicals are known to quench fluorophores upon 'contract', the results reflect the relative interaction of local anesthetics with membrane lipids and proteins. The evidence is consistent with the concept of multiple binding sites for local anesthetics in membranes. Local anesthetics, once intercalated into the bilayer, may diffuse laterally and interact with membrane components, lipid as well as proteins. In biological membranes, however, positively charged local anesthetics are better able to quench 1-anilinonaphthalene 8-sulfonate in protein regions, suggesting that the interaction between local anesthetics and membrane proteins can be electrostatic in nature.     

Fluidizing the membrane by a local anesthetic: phenylethanol affects membrane protein oligomerization

The exact mechanism of action of anesthetics is still an open question. While some observations suggest specific anesthetic-protein interactions, nonspecific perturbation of the lipid bilayer has also been suggested. Perturbations of bilayer properties could subsequently affect the structure and function of membrane proteins. Addition of the local anesthetic phenylethanol (PEtOH) to model membranes and intact Escherichia coli cells not only affected membrane fluidity but also severely altered the defined helix-helix interaction within the membrane. This experimental observation suggests that certain anesthetics modulate membrane physical properties and thereby indirectly affect transmembrane (TM) helix-helix interactions, which are not only involved in membrane protein folding and assembly but also important for TM signaling.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.     

NMR study of volatile anesthetic binding to nicotinic acetylcholine receptors

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using (19)F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K(d), of isoflurane binding to nAChR at 15 degrees C was found to be 0.36 +/- 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X(b), determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T(2)) of (19)F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k(-1), is 1.7 x 10(4) s(-1). The on rate, k(+1), can thus be calculated to be approximately 4.8 x 10(7) M(-1) s(-1), suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k(+1) and k(-1) are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.     

Porphyrin-membrane interactions: binding or partition?

Porphyrins are photodynamic drugs employed in an experimental tumor-treatment modality in which cell membranes are one of the primary drug-action sites. To gain insight into the nature of the interaction of these drugs with those primary sites we have studied the affinity of porphyrins to the lipid moieties of biological membranes, at the molecular level. The association of porphyrins to large unilamellar liposomes, modeling the lipid regions of biological membranes was studied (at equilibrium) for deuteroporphyrin IX and protoporphyrin IX, at neutral pH and 37 degrees C, taking into account porphyrin aggregation. Two thermodynamic approaches were investigated: (i) Simple partition equilibria between the external aqueous phase and the lipid bilayer, for drug monomers and dimers. (ii) Binding equilibria of drug monomers and dimers to the lipid bilayer. Using two types of experimental design and processing the data according to the expectations of both approaches, three different models for the binding (differing in the participation assigned to the dimer) were considered. Our major findings are: (a) The data clearly do not fit with the expectations for simple partition equilibria, nor with binding models assuming direct participation of the dimers. (b) The data fit well with a binding process, in which the membrane binds the porphyrin monomers only, with the dimers participating indirectly through the aqueous dimerization equilibrium. (c) At 37 degrees C and neutral pH, for liposomes composed of phosphatidylcholine/cholesterol at molar ratios of 3:2, we found for both investigated species a binding constant of 2.3 x 10(4) M-1. (d) For each species the binding constant is independent of the initial and final states of drug aggregation in the aqueous phase.     

Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.     

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mechanism of general anesthesia: III. Kinetic studies on erythrocyte ghost acetylcholinesterase

General anesthetics inhibit erythrocyte membrane-bound acetylcholinesterase. Release of the membrane-bound enzyme by sonication into a soluble form induces a loss of sensitivity to anesthetics. Reconstitution of the solubilized enzyme with phospholipids restores its inhibition by anesthetics. The results suggest that anesthetic inhibition of acetylcholinesterase is mediated through the lipid bilayer.     

The effect of a myelin apoprotein on lipid structure: a spin probe study.

Spin probes, stable free radical derivatives of stearic acid and cholestanone, were used to observe the effects of the "Folch-Lees" protein isolated from the white matter of bovine brain on the organization and motion of lipid molecules. The incorporation of the organic solvent soluble form of this protein decreased the tendency of a variety of lipid molecules with zero, positive or negative net charges to arrange themselves close to the normal to the lipid bilayer. The aqueous form of the protein also had a profound chaotropic effect on the molecular geometry of the lipid, but only if the lipids had a net negative charge (the protein has a net positive charge in the pH range investigated). Examination of the ESR spectra indicated that this protein altered the geometry of the lipid structure without causing major changes in the mobility of the individual lipid molecules.     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.     

Concentration effects of volatile anesthetics on the properties of model membranes: a coarse-grain approach

To gain insights into the molecular level mechanism of drug action at the membrane site, we have carried out extensive molecular dynamics simulations of a model membrane in the presence of a volatile anesthetic using a coarse-grain model. Six different anesthetic (halothane)/lipid (dimyristoylphosphatidylcholine) ratios have been investigated, going beyond the low doses typical of medical applications. The volatile anesthetics were introduced into a preassembled fully hydrated 512-molecule lipid bilayer and each of the molecular dynamics simulations were carried out at ambient conditions, using the NPT ensemble. The area per lipid increases monotonically with the halothane concentration and the lamellar spacing decreases, whereas the lipid bilayer thickness shows no appreciable differences and only a slight increase upon addition of halothane. The density profiles of the anesthetic molecules display a bimodal distribution along the membrane normal with maxima located close to the lipid-water interface region. We have studied how halothane molecules fluctuate between the two maxima of the bimodal distribution and we observed a different mechanism at low and high anesthetic concentrations. Through the investigation of the reorientational motions of the lipid tails, we found that the anesthetic molecules increase the segmental order of the lipids close to the membrane surface.