Alcohol's effects on lipid bilayer properties

Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol''s biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1–16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket.     

Conductivity and structural transitions in bilayer lipid membranes

5 structural transitions were found in bilayer lipid membranes (BLM) from egg lecithin (EL) within the temperature range 14-44 degrees C. In the transition zone BLM conductivity abruptly increases, in some cases current fluctuations of the order 150 pC of the channel type are initiated. The transition temperatures observed in BLM from EL coincide with those in biological membranes. The cause of this phenomenon is discussed, as well as possible use of these BLM in the region of structural transition as a model of cellular receptor to electromagnetic fields.     

Glycine receptors: recent insights into their structural organization and functional diversity

Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.     

AFM study on the electric-field effects on supported bilayer lipid membranes

Electric-field induced changes in structure and conductivity of supported bilayer lipid membranes (SLM) have been studied at submicroscopic resolution using atomic force microscopy and electrochemical impedance spectroscopy. The SLMs are formed on gold surfaces modified with mixed self-assembled monolayers of a cholesterol-tether and 6-mercaptohexanol. At applied potentials of ≤−0.25 V versus standard hydrogen electrode, the conductance of the SLM increases and membrane areas of <150 nm in size are found to elevate from the surface up to 15 nm in height. To estimate the electric field experienced by the lipid membrane, electrowetting has been used to determine the point of zero charge of a 6-mercaptohexanol-modified surface (0.19 ± 0.13 V versus standard hydrogen electrode). The effects of electric fields on the structure and conductance of supported membranes are discussed.     

Membrane topology of insulin receptors reconstituted into lipid vesicles

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the - and the -subunit of the receptor. Immunogold labeling of the C-terminus of the -subunit indicates that it resides about 6 nm off the membrane, while two -subunit epitopes were labeled at about twice this distance, confirming that the -subunit is harbored in the cross-bar of the T-structure.We thank Ms. Birthe Nystrøm, Lisette Hansen and Ulla Blankensteiner for excellent technical assistance and Ms. Birgit Risto for skillful work with the photographic prints.     

The formation and annealing of structural defects in lipid bilayer vesicles

It is shown that sonication of phospholipid-water dispersions below the crystalline → liquid crystalline phase transition temperature (T_c) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above T_c. The rate of annealing was found to be slow, of the order of an hour for T = 3 °C above T_c, but annealing is complete within 10 min for T = 10 °C above T_c. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside ? outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 °C above the phase transition.     

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Chemical and solvent effects on the interaction of tetraphenylborate with lipid bilayer membranes

Alkali metal salts of tetraphenylboron dissociate in aqueous solution to yield the hydrophobic anion, BPh₄^?, which is strongly adsorbed at the surfaces of lipid bilayer membranes. Upon application of a transmembrane voltage pulse these anions cross the membrane without appreciable desorption, thereby exhibiting a transient electrical conductance. The relaxation time of this transient is governed by the height of the central potential barrier which the anions must surmount in crossing the membrane. Because of discrete charge effects, the barrier height and hence the observed relaxation time increase markedly with increasing surface density of adsorbed BPh₄^?. Since adsorbed BPh₄^? are in partition equilibrium with the same species dissolved in the aqueous phase, measurement of the relaxation time for BPh₄^? membrane conductance can be used to assay the aqueous-phase concentration of the hydrophobic anion. In this way we have observed the precipitation of KBPh₄ in water, obtaining a solubility concentration product of 1.0·10^?7 mol²·dm^?6 for the precipitation reaction at 25°C. This result is larger by a factor of two than the most directly comparable published values from other sources. In additional experiments we have reduced the polarity of the aqueous phases bathing the membrane by adding varying amount of ethylene glycol to the water. Using the same conductance relaxation assay, we have determined that partitioning of BPh₄^? into the membrane/solution interfaces is lessened as the polarity of the bathing solutions is reduced. This result is attributed to a lowering of the chemical potential of the BPh₄^? in the less polar medium.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

Solute effects on the colloidal and phase behavior of lipid bilayer membranes: ethanol-dipalmitoylphosphatidylcholine mixtures.

By means of the scanning differential calorimetry, x-ray diffractometry, and the dynamic light scattering, we have systematically studied the phase and packing properties of dipalmitoylphosphatidylcholine vesicles or multibilayers in the presence of ethanol. We have also determined the partial ternary phase diagram of such dipalmitoylphosphatidylcholine/water/ethanol mixtures. The directly measured variability of the structural bilayer parameters implies that ethanol binding to the phospholipid bilayers increases the lateral as well as the transverse repulsion between the lipid molecules. This enlarges the hydrocarbon tilt (by up to 23 degrees) and molecular area (by < or = 40%). Ethanol-phospholid association also broadens the interface and, thus, promotes lipid headgroup solvation. This results in excessive swelling (by 130%) of the phosphatidylcholine bilayers in aqueous ethanol solutions. Lateral bilayer expansion, moreover, provokes a successive interdigitation of the hydrocarbon chains in the systems with bulk ethanol concentrations of 0.4-1.2 M. The hydrocarbon packing density as well as the propensity for the formation of lamellar gel phases simultaneously increase. The pretransition temperature of phosphatidylcholine bilayers is more sensitive to the addition of alcohol (initial shift: delta Tp = 22 degrees C/mol) than the subtransition temperature (delta Ts reversible 5 degrees C/mol), whereas the chain-melting phase transition temperature is even less affected (delta Tm = 1.8 degrees C/mol). After an initial decrease of 3 degrees for the bulk ethanol concentrations below 1.2 M, the Tm value increases by 2.5 degrees above this limiting concentration. The gel-phase phosphatidylcholine membranes below Tm are fully interdigitated above this limiting concentration. The chain tilt on the fringe of full chain interdigitation is zero and increases with higher ethanol concentrations. Above Tm, some of the lipid molecules are solubilized by the bound ethanol molecules. More highly concentrated ethanol solutions (> 7 M) solubilize the phosphatidylcholine bilayers with fluid chains fully and result in the formation of mixed lipid-alcohol micelles.     

Different effects of di- and triphenyltin compounds on lipid bilayer dithionite permeabilization

Phenyltins are chemicals widely used in industry, hence their occurrence in the human environment is frequent and widespread. Such compounds include hydrophobic phenyl rings bonded to positively charged tin. This molecular structure makes them capable of adsorbing onto and penetrating through biological membranes, hence they are potentially hazardous. Two such compounds, diphenyltin and triphenyltin, show different steric constraints when interacting with the lipid bilayer. It has been demonstrated that these compounds are positioned at different locations within model lipid bilayers, causing dissimilarity in their ability to affect membrane properties. In this paper we present a study regarding the ability of these two phenyltins to facilitate the transport of S2O4(-2) ions across the lipid bilayer, evaluated by a fluorescence quenching assay. In concentration range of up-to 60 microM those compounds do not affect lipid bilayer topology, when evaluated by vesicle size distribution. Both phenyltins facilitate the transfer of S2O4(-2) across the model lipid bilayer, but the dependence of dithionite transport on phenyltin concentration is different for both. In principle, above 20 microM triphenyltin is more efficient in transferring ions across the lipid bilayer than diphenyltin.     

Average structural and motional properties of a diunsaturated acyl chain in a lipid bilayer: effects of two cis-unsaturated double bonds

Isolinoleic acid (18:2 delta 6,9) deuterated at 10 different positions was esterified to form 1-palmitoyl-2-isolinoleoyl-sn-glycero-3-phosphocholine (PiLPC), and the average structural and motional properties of the diunsaturated chain, in aqueous dispersions of PiLPC, were examined by 2H NMR spectroscopy. For each sample, 2H spectra were acquired over a temperature range of 1-40 degrees C and the quadrupolar splittings interpreted in terms of carbon-deuterium bond order parameters, SCD. Furthermore, definition of the average orientation of the C8 methylene unit with respect to the bilayer normal [Baenziger, J. E., Smith, I. C. P., Hill, R. J., & Jarrell, H. C. (1988) J. Am. Chem. Soc. 110, 8229-8231] provided sufficient information to calculate both the average orientations and the molecular order parameters, Smol (which reflects the amplitudes of motion), for the C6-C7 and the C9-C10 double bonds. The results indicate that both the motional freedom (reflected in the order profile) and the average structure (reflected in the orientation of carbon segments with respect to the bilayer normal) are strongly affected by the presence of two cis-unsaturated double bonds. The data were interpreted in terms of two possible models whereby, in each case, the chain adopts a conformation consistent with the low-energy conformation of 1,4-pentadiene [Applegate, K. R., & Glomset, J. A. (1986) J. Lipid Res. 27, 658-680] but undergoes a two-site jump between the conformations. The jump motion arises mainly from rotations about the C7-C8 and the C8-C9 single bonds that disorder the C8 and the C9-C10 segments (Smol = 0.15 and 0.08, respectively) but leave the C6-C7 double bond relatively immobile (Smol = 0.55; all at 40 degrees C). It is suggested that acyl chains containing three or more double bonds could not undergo a similar jump motion and therefore would be highly ordered and not "fluid" as is generally thought.     

Dynamical and structural properties of charged and uncharged lidocaine in a lipid bilayer

Molecular dynamics computer simulations have been performed to investigate dynamical and structural properties of a lidocaine local anesthetic. Both charged and uncharged forms of the lidocaine molecule were investigated. Properties such as membrane area per lipid, diffusion, mass density, bilayer penetration and order parameters have been examined. An analysis of the lidocaine interaction with the lipid surrounding according to a simple mean field theory has also been performed. Almost all examined properties were found to depend on which of the two forms of lidocaine, charged or uncharged, is studied. The overall picture is a rather static behavior determined by the lipids for the charged molecules and more mobile situation of the uncharged form with higher diffusion and lower orientational and positional order.     

Location and orientation of DODCI in lipid bilayer membranes: effects of lipid chain length and unsaturation.

The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12>C14>C16>C18C20) and in unsaturated lipids (C14:1>C14:0, C16:1>C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule in the interior region of the membrane is more when the structure of th bilayer is more fluid-like.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Direct imaging of salt effects on lipid bilayer ordering at sub-molecular resolution

The interactions of salts with lipid bilayers are known to alter the properties of membranes and therefore influence their structure and dynamics. Sodium and calcium cations penetrate deeply into the headgroup region and bind to the lipids, whereas potassium ions only loosely associate with lipid molecules and mostly remain outside of the headgroup region. We investigated a dipalmitoylphosphatidylcholine (DPPC) bilayer in the gel phase in the presence of all three cations with a concentration of Ca²⁺ ions an order of magnitude smaller than the Na⁺ and K⁺ ions. Our findings indicate that the area per unit cell does not significantly change in these three salt solutions. However the lipid molecules do re-order non-isotropically under the influence of the three different cations. We attribute this reordering to a change in the highly directional intermolecular interactions caused by a variation in the dipole-dipole bonding arising from a tilt of the headgroup out of the membrane plane. Measurements in different NaCl concentrations also show a non-isotropic re-ordering of the lipid molecules.     

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.     

Alcohol effects on lipid bilayer permeability to protons and potassium: relation to the action of general anesthetics

Past work has shown that general anesthetics perturb the membranes of isolated synaptic vesicles, thereby increasing permeability to protons and inhibiting the ability of the vesicles to take up catecholamines. It has been proposed that such effects may produce anesthesia through inhibition of synaptic transmission. The mechanisms of perturbation is unknown. Two possible explanations include alterations of dielectric constant or production of defects as anesthetics partition into the bilayer phase. In order to choose between these alternatives, we measured the effect of nine alcohols and two alkanes on liposome permeability to protons and potassium. Ionic permeability was increased by alcohols and alkanes to similar degrees, thereby ruling out direct effects on the membrane dielectric constant caused by partitioning of anesthetics into the bilayer. Other experiments confirmed earlier reports that the enhanced permeability caused by anesthetics is not specific for protons. We conclude that these membrane perturbants act by increasing the number of transient, ion-conducting defects normally present in the bilayer structure.