Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.     

Membrane localization and dynamics of Nile Red: effect of cholesterol

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.     

Membrane localization and dynamics of Nile Red: Effect of cholesterol

The organization and dynamics of the hydrophobic fluorescent probe Nile Red incorporated in DOPC vesicles containing varying amounts of cholesterol has been monitored utilizing fluorescence-based approaches which include the red edge excitation shift (REES) approach and the parallax method for depth determination. Our results show that the fluorescence emission maximum, intensity, polarization, and lifetime of Nile Red vary with the cholesterol content of the membrane. Interestingly, Nile Red exhibits significant REES independent of the presence of cholesterol. This indicates that Nile Red is localized in a motionally restricted environment in the membrane. This is supported by analysis of membrane penetration depth of Nile Red using the parallax method which points out to a membrane interfacial localization of Nile Red. These results could be useful in analyzing membrane organization and heterogeneity in natural membranes using Nile Red.     

Cholesterol-induced changes in hippocampal membranes utilizing a phase-sensitive fluorescence probe

The function of membrane receptors in the nervous system depends on physicochemical characteristics of neuronal membranes such as membrane order and phase. In this work, we have monitored the changes in hippocampal membrane order and related parameters by cholesterol and protein content utilizing a Nile Red-based phase-sensitive fluorescent membrane probe NR12S. Since alteration of membrane cholesterol is often associated with membrane phase change, the phase-sensitive nature of NR12S fluorescence becomes useful in these experiments. Our results show that fluorescence spectroscopic parameters such as emission maximum, anisotropy, and lifetime of NR12S display characteristic dependence on membrane cholesterol content. Interestingly, cholesterol-dependent red edge excitation shift is displayed by NR12S under these conditions. Hippocampal membranes exhibited reduction in liquid-ordered phase upon cholesterol depletion. These results provide insight into changes in hippocampal membrane order in the overall context of cholesterol and protein modulation.     

Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study

Lipid rafts, the functional microdomains in the cell membrane, are believed to exist as liquid-ordered (Lo) phase domains along with the liquid-disordered (Ld) phase of the bulk of the cell membranes. We have examined the lipid order in model and natural membranes by time-resolved fluorescence of trimethylammonium-1,6-diphenylhexatriene incorporated into the membranes. The lipid phases were discerned by the limiting anisotropy, rotational diffusion rate and distribution of the fluorescence lifetime. In dipalmitoylphosphatidylcholine (DPPC)-cholesterol mixtures the gel phase exhibited higher anisotropy and a two-fold slower rotational diffusion rate of the probe as compared to the Ld phase. On the other hand, the Lo phase exhibited higher limiting anisotropy but a rotational diffusion rate comparable to the Ld phase. The Ld and Lo phases elicited unimodal distribution of lifetimes with distinct mean values and their co-existence in phospholipid-cholesterol mixtures was reflected as a biphasic change in the width of the lifetime distribution. Global analysis of the lifetimes yielded a best fit with two lifetimes which were identical to those observed in single Lo or Ld phases, but their fractional contribution varied with cholesterol concentration. Attributing the shorter and longer lifetime components to the Ld and Lo phases, respectively, the extent of the Lo/Ld phase domains in the membranes was estimated by their fractional contribution to the fluorescence decay. In ternary mixtures of egg PC-gangliosides-cholesterol, the gangliosides induced heterogeneity in the membrane but the Ld phase prevailed. The Lo phase properties were observed only in the presence of cholesterol. Results obtained in the plasma membrane and detergent-resistant membrane fractions (DRMs) isolated from U-87 MG cells revealed that DRMs mainly possess the Lo phase; however, a substantially large proportion of plasma membrane also exists in the Lo phase. Our data show that, besides cholesterol, the membrane proteins play a significant role in the organization of lipid rafts and, furthermore, a considerable amount of heterogeneity is present among the lipid rafts.     

Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes

Cholesterol is an abundant lipid of mammalian membranes and plays a crucial role in membrane organization, dynamics, function and sorting. The role of cholesterol in membrane organization has been a subject of intense investigation that has largely been carried out in model membrane systems. An extension of these studies in natural membranes, more importantly in neuronal membranes, is important to establish a relationship between disease states and changes in membrane physical properties resulting from an alteration in lipid composition. We have monitored the lateral diffusion of lipid probes, DiIC(18)(3) and FAST DiI which are similar in their intrinsic fluorescence properties but differ in their structure, in native and cholesterol-depleted hippocampal membranes using the fluorescence recovery after photobleaching (FRAP) approach. Our results show that the mobility of these probes is in general higher in hippocampal membranes depleted of cholesterol. Interestingly, the increase in mobility of these probes does not linearly correlate with the extent of cholesterol depletion. These results assume significance in the light of recent reports on the requirement of cholesterol to support the function of the G-protein coupled serotonin(1A) receptor present endogenously in hippocampal membranes.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin1A receptor: Implications in desmosterolosis

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin_1A receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin_1A receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.     

Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.     

PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

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.     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers

A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, and cholesterol content. Probe F, due to excited-state intramolecular proton transfer, exhibits two bands in emission that are differently sensitive to intermolecular interactions-thereby allowing us to distinguish universal (dipole-dipole) and specific (H-bonding) interactions within the bilayer. Spectroscopic, quenching, and anisotropy data suggest the presence of two forms of probe F at different locations in the bilayer: an H-bond free form located below sn(1)-carbonyls and an H-bonded form located at the polar membrane interface. We provide a quantitative analysis of the distribution of the probe between these two locations as well as the polarity of these locations, and show that both the distribution and the polarity contribute to the probe response. Moreover, analysis of literature data on other environment-sensitive probes (Prodan, Laurdan, Nile Red, NBD lipids, etc.) in lipid bilayers allows us to suggest that the bimodal distribution in the lipid bilayer is probably a general feature of low-polar molecules with polar groups capable of H-bonding interactions.     

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.     

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside,GM1

Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM₁) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λ_max) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λ_max in the presence and absence of two important components of neuronal membranes, cholesterol and GM₁ indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.