Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

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.     

Lipid peroxidation-induced changes in physical properties of annular lipids in rat brain synaptosomal membranes

The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.     

Effect of the anesthetics benzyl alcohol and chloroform on bilayers made from monolayers.

The neutral anesthetics chloroform and benzyl alcohol, at concentrations that block the nerve impulse, greatly modify the transport parameters of positive and negative ions in lipid bilayers made from monolayers. Both chloroform and benzyl alcohol increase the membrane permeability to these ions and increase the translocation rate for tetraphenylborate. It was found that both anesthetics increase the membrane permeability to positive ions more markedly than to negative ions. It was also found that the membrane capacitance increases lineary with the concentration of benzyl alcohol. At 51 mM benzyl alcohol, the increase in capacitance is approximately 6%. Chloroform also increases the membrane capacitance; the increase in capacitance was found to be 6% at 18 mM chloroform. An analysis of the changes in the transport parameters of the lipophilic ions, together with the changes in membrane capacitance, suggests that benzyl alcohol and chloroform modify the dipole potential and dielectric constant of the membrane. Benzyl alcohol may also increase the "fluidity" of the lipid bilayer membranes. At 36 mM benzyl alcohol, the membrane permeability to acetamide increases by 38%.     

Bilayer Thickness and Curvature Influence Binding and Insertion of a pHLIP Peptide

The physical properties of lipid bilayers, such as curvature and fluidity, can affect the interactions of polypeptides with membranes, influencing biological events. Additionally, given the growing interest in peptide-based therapeutics, understanding the influence of membrane properties on membrane-associated peptides has potential utility. pH low insertion peptides (pHLIPs) are a family of water-soluble peptides that can insert across cell membranes in a pH-dependent manner, enabling the use of pH to follow peptide-lipid interactions. Here we study pHLIP interactions with liposomes varying in size and composition, to determine the influence of several key membrane physical properties. We find that pHLIP binding to bilayer surfaces at neutral pH is governed by the ease of access to the membrane’s hydrophobic core, which can be facilitated by membrane curvature, thickness, and the cholesterol content of the membrane. After surface binding, if the pH is lowered, the kinetics of pHLIP folding to form a helix and subsequent insertion across the membrane depends on the fluidity and energetic dynamics of the membrane. We showed that pHLIP is capable of forming a helix across lipid bilayers of different thicknesses at low pH. However, the kinetics of the slow phase of insertion corresponding to the translocation of C-terminal end of the peptide across lipid bilayer, vary approximately twofold, and correlate with bilayer thickness and fluidity. Although these influences are not large, local curvature variations in membranes of different fluidity could selectively influence surface binding in mixed cell populations.     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

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.     

Membrane actions of water-soluble fusogens: Effect of dimethyl sulfoxide,glycerol and sucrose on lipid bilayer order and fluidity

The effect of three water-soluble fusogens: dimethyl sulfoxide (DMSO), glycerol and sucrose on the structural properties of model lipid membranes has been studied by electron spin resonance (ESR) using 5-doxylstearic acid as a spin probe and by fluorescence spectroscopy using pyrene as an excimer forming fluorescent probe. All three fusogens tested produce a marked increase in the order parameter of the region close to the polar surface of the lipid bilayer. The ordering effect of DMSO, but not of glycerol and sucrose, is much stronger with respect to membranes prepared from acidic than from neutral phospholipids. The membrane-perturbing action of glycerol and sucrose manifests itself also in the reduced lateral mobility of membrane incorporated pyrene, indicating thus a decreased fluidity of the bilayer hydrophobic region. The structural perturbations produced in model membranes by DMSO, glycerol and sucrose are discussed in relation to the mechanism by which these substances promote cell fusion.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

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.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Permeability of lipid membranes revised in relation to freeze-thaw processes.

Water and solute activity gradients created during freeze-thaw processes produce water and solute fluxes across the cell membrane resulting in volume changes. Under these conditions, osmotic and thermal stresses affect the curvature, the phase behavior, and the surface properties of the lipid bilayer. These structural changes are not considered by the classical formalisms describing permeability of lipid membranes to water and nonelectrolytes such as the Nernst-Planck equation, Eyring's absolute rate theory, and Kedem-Katchalsky's thermodynamic of irreversible processes approach. In this paper, the influence of such changes on the glycerol permeation kinetics are reported. The results indicate that osmotic and chemical effects of the cryoprotectant on the membrane properties affect the rate of volume swelling depending on whether the membrane is in the gel or in the liquid crystalline state.     

The effect of membrane-fluidizing agents on the adhesion of CHO cells

Treatment of CHO cells with drugs which are known to increase membrane lipid fluidity reduced the cells' ability to adhere to protein coated substrates, The concentrations of local anesthetics, nonionic detergents or aliphatic alcohols required to reduce CHO cell adhesion by 50% were similar to those reported to block nerve conduction, indicating that these drugs can affect the membrane at physiologically significant concentrations. Nonionic detergents and aliphatic alcohols, but not local anesthetics, caused increases in the fluidity of CHO plasma membranes (measured by fluorescence polarization) at concentrations which inhibited cell adhesion. The adhesion versus temperature profile had a sigmoidal shape, suggesting that a temperature dependent cooperative process such as a lipid phase transition, might be involved. However, the temperature profile for CHO membrane fluidity manifested no discontinuities, indicating the absence of any discrete phase transitions of the lipid matrix. This observation, coupled with the result that the inhibition of CHO cell adhesion produced by low temperatures was not relieved by drugs which can increase membrane fluidity, suggests that the reduced adhesion seen at low temperature is probably not due to reduced lipid fluidity.     

Role of leaflet asymmetry in the permeability of model biological membranes to protons, solutes, and gases.

Bilayer asymmetry in the apical membrane may be important to the barrier function exhibited by epithelia in the stomach, kidney, and bladder. Previously, we showed that reduced fluidity of a single bilayer leaflet reduced water permeability of the bilayer, and in this study we examine the effect of bilayer asymmetry on permeation of nonelectrolytes, gases, and protons. Bilayer asymmetry was induced in dipalmitoylphosphatidylcholine liposomes by rigidifying the outer leaflet with the rare earth metal, praseodymium (Pr3+). Rigidification was demonstrated by fluorescence anisotropy over a range of temperatures from 24 to 50 degrees C. Pr3+-treatment reduced membrane fluidity at temperatures above 40 degrees C (the phase-transition temperature). Increased fluidity exhibited by dipalmitoylphosphatidylcholine liposomes at 40 degrees C occurred at temperatures 1-3 degrees C higher in Pr3+-treated liposomes, and for both control and Pr3+-treated liposomes permeability coefficients were approximately two orders of magnitude higher at 48 degrees than at 24 degrees C. Reduced fluidity of one leaflet correlated with significantly reduced permeabilities to urea, glycerol, formamide, acetamide, and NH3. Proton permeability of dipalmitoylphosphatidylcholine liposomes was only fourfold higher at 48 degrees than at 24 degrees C, indicating a weak dependence on membrane fluidity, and this increase was abolished by Pr3+. CO2 permeability was unaffected by temperature. We conclude: (a) that decreasing membrane fluidity in a single leaflet is sufficient to reduce overall membrane permeability to solutes and NH3, suggesting that leaflets in a bilayer offer independent resistances to permeation, (b) bilayer asymmetry is a mechanism by which barrier epithelia can reduce permeability, and (c) CO(2) permeation through membranes occurs by a mechanism that is not dependent on fluidity.     

The evaluation of the age-related characteristics of the structural status of the plasma membrane lipid phase in rat fatty tissue by using the method of inductive-resonance energy transfer

Using the method of inductance-resonance energy transfer from tryptophanyl residues to fluorescent pyrene probe the structural state of plasmatic membranes from adipose tissue of different age rats has been studied. The structural heterogeneity of membrane lipid phase has been revealed. The differences in physical properties of annular and bilayer lipids don't depend on age. During aging the membrane lipid viscosity including lipids of near protein area decreases, the conformation of membrane protein components alters during aging as well. The data on various effectiveness of energy transfer from tryptophanyls to pyrene probe in young and aged animals with stable polypeptide composition of membrane proteins indicates that. The structure of membrane lipid phase is suggested to be the main factor affecting the conformational state and functional activity of membrane-bound proteins during aging.     

The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.