The interaction of antipsychotic drugs with lipids and subsequent lipid reorganization investigated using biophysical methods

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands.     

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025–0.15 mM; dodecyl sulphate 0.82 in the range 0.01–1 mM; sodium deoxycholate 1.03 in the range 0.01–1 mM and sodium cholate 1.37 in the range 0.1–10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.     

The lipid dependence of melittin action investigated by dual-color fluorescence burst analysis

Dual-color fluorescence-burst analysis was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform dual-color fluorescence-burst analysis, fluorescently labeled size-marker molecules were encapsulated into liposomes, labeled with a second lipid-attached fluorophore. By correlating the fluorescence bursts, resulting from the liposomes diffusing through the detection volume of a dual-color confocal microscope, the distribution of size-marker molecules over the liposomes was determined. It was found that melittin causes leakage via two different mechanisms: 1), For liposomes composed of neutral bilayer-forming lipids, low melittin concentrations induced pore formation with the pore size depending on the melittin concentration. 2), For liposomes containing anionic and/or nonbilayer forming lipids, melittin induced fusion or aggregation of liposomes accompanied by a-specific leakage. Experiments with liposomes prepared from Escherichia coli lipid extracts and intact cells of Lactococcus lactis indicate that both mechanisms are physiologically relevant.     

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.     

The interaction of hydrophobic ions with lipid bilayer membranes

Summary Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and Läuger (J. Membrane Biol. 5:225, 1971). This model visualizes as three distinct steps the interfacial adsorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions adsorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon adsorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon adsorption, of a highly ordered water structure which surrounds the hydrophobic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead be attributed to a more complex effect equivalent to a reduction of membrane fluidity.Research performed while on sabbatical leave April-September, 1974.     

The interaction of bee melittin with lipid bilayer membranes

The influence of melittin and the related 8-26 peptide on the stability and electrical properties of bilayer lipid membranes is reported. Melittin, unlike the 8-26 peptide, has a dramatic influence on lipid membranes, causing rupture at dilute concentrations. The circular dichroism of melittin demonstrated that under physiological conditions, in water, melittin is in extended conformation, which is enhanced in aqueous ethanol. However in 'membrane-like' conditions it is essentially alpha-helical. Secondary structure predictions were used to locate possible alpha-helical nucleation centres and a model of melittin was built according to these predictions. It is postulated that melittin causes a wedge effect in membranes.     

The interaction of anionic detergents with lipid bilayer membranes

Summary The time course of the reaction of anionic surfactants with lipid bilayers is followed and analyzed. The distribution of detergents in the membrane phase gives rise to an asymmetry potential followed by a diffusion potential. Detergent-doped membranes are cation-permselective. It is postulated that a variable profile of mobile charges in the membrane account for the cation-permselectivity, the intercation selectivity, and the voltage-dependent gating phenomena observed in excitable membranes.Supported by a grant from the Medical Research Council of Canada.I thank Mr. G. Beauchesme for his technical assistance in part of this work.     

The interaction of viral fusion peptides with lipid membranes

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.     

pH-dependent effects of local anaesthetics in perturbing lipid membranes

The effects of local anaesthetics lidocaine, benzocaine, carbisocaine and carbisocaine derivatives, KaQ-7 and Ka-O, in perturbing bovine brain lipid membranes or egg lecithin membranes were compared at pH 6.0; 7.0; and 8.0. The electron spin resonance method with stearic acid labeled at carbon position 16 as the spin probe was employed. The perturbation effects of lidocaine and Ka-O were found to increase with increasing pH of the sample, whereas the effect of carbisocaine decreased with increasing pH. The perturbation effects of benzocaine and KaQ-7 were independent of pH. The pH-dependent perturbation effects of the local anaesthetics tested on lipid membrane fairly corresponded with their pH-dependent potency to block nerve action potentials.     

The interaction of hydrophobic ions with lipid bilayer membranes.

Electrical relaxation studies have been made on lecithin bilayer membranes of varying chain length and degree of unsaturation, in the presence of dipicrylamine. Results obtained are generally consistent with a model for the transport of hydrophobic ions previously proposed by Ketterer, Neumcke, and L?uger (J. Membrane Biol. 5:225, 1971). This medel visualizes as three distinct steps the interfacial absorption, translocation, and desorption of ions. Measurements at high electric field yield directly the density of ions absorbed to the membrane-solution interface. Variation of temperature has permitted determination of activation enthalpies for the translocation step which are consistent with the assumption of an electrostatic barrier in the hydrocarbon core of the membrane. The change of enthalpy upon absorption of ions is, however, found to be negligible, the process being driven instead by an increase of entropy. It is suggested that this increase may be due to the destruction, upon absorption, of a highly ordered water structure which surrounds the hydrophic ion in the aqueous phase. Finally, it is shown that a decrease of transient membrane conductance observed at high concentration of hydrophobic ions, previously interpreted in terms of interfacial saturation, must instead by attributed to a more complex effect equivalent to a reduction of membrane fluidity.     

On the interaction of hemocyanin with lipid bilayer membranes

Osmotic jump experiments were used to measure the ionic permeability induced in lipid vesicles by Megathura crenulata hemocyanin. It was found that this protein strongly increases the conductance of K⁺ and Cl^- through these membranes but not that of SO ₄ ⁼ . These effects were attributed to the formation of ionic channels in the vesicles. We have found that a simple first-order binding model can explain the dependence of the number of pore-containing vesicles both on the time after exposure to hemocyanin and on the protein concentration. Milder effects were attributed to a non-specific adhesion of the protein to the membrane surface. Consistent with the hypothesis of reversible association, vesicles which retained hemocyanin after step sucrose density gradient centrifugation at low ionic strength, lost most of the protein upon recentrifugation at high ionic strength. Consistent with the hypothesis of channel formation bot the above vesicle preparations transferred voltage-dependent hemocyanin channels into planar bilayers when they were made to fuse with them. It is concluded that hemocyanin can interact both specifically, by forming pores within the hydrophobic core of lipid membranes, and non-specifically, probably by means of electrostatic interaction with the surface of the same membrane.Abbreviations Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - DOC sodium deoxycholate     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm² for DPhyPC bilayers and 0.75 and 0.77 μF/cm² for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm² were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Prion protein structure is affected by pH-dependent interaction with membranes: a study in a model system

Interaction of full length recombinant hamster prion protein with liposomes mimicking lipid rafts or non-raft membrane regions was studied by circular dichroism, chemical cross-linking and sucrose gradient ultracentrifugation. At pH 7.0, the protein bound palmitoyloleoylphosphatidylcholine/cholesterol/sphingomyelin/monosialoganglioside GM1 (GM1) ganglioside liposomes but not palmitoyloleoylphosphatidylcholine alone (bound/free=0.33 and 0.01, respectively), maintaining the native alpha-helical structure and monomeric form. At pH 5.0, though still binding to quaternary mixtures, in particular GM1, the protein bound also to palmitoyloleoylphosphatidylcholine (bound/free 0.35) becoming unfolded and oligomeric. The pH-dependent interaction with raft or non-raft membranes might have implication in vivo, by stabilizing or destabilizing the protein.     

The interaction between lipid derivatives of colchicine and tubulin: consequences of the interaction of the alkaloid with lipid membranes

Colchicine is a potent antimitotic poison which is well known to prevent microtubule assembly by binding tubulin very tightly. Colchicine also possesses anti-inflammatory properties which are not well understood yet. Here we show that colchicine tightly interacts with lipid layers. The physical and biological properties of three different lipid derivatives of colchicine are investigated parallel to those of membrane lipids in the presence of colchicine. Upon insertion in the fatty alkyl chains, colchicine rigidifies the lipid monolayers in a fluid phase and fluidifies rigid monolayers. Similarly X-ray diffraction data show that lecithin-water phases are destabilized by colchicine. In addition, an unexpectedly drastic enhancement of the photoisomerization rate of colchicine into lumicolchicine in the lipid environment is observed and further supports insertion of the alkaloid in membranes. Finally the interaction of colchicine with lipids makes the drug inaccessible to tubulin. The possible in vivo significance of these results is discussed.