The Kupershtokh-Medvedev electrostrictive instability as possible mechanism of initiation of phase transitions, domains and pores in lipid membranes and influence of microwave irradiation on cell

One of the possible mechanisms of initiation of local phase transitions and formation of nonuniform structure of biological and model lipid membranes is suggested. It is based on anisotropic electrohydrodynamic instability of Kupershtokh and Medvedev in strong electric field relative to density perturbations. This mechanism may clarify initial stages of formation of membrane domains and pores, some aspects of cell signalization and influence of microwave irradiation of nonthermal intensity on living organisms.     

"Blocking" by polyethyleneglycol of single lipid pores arising in unmodified bilayer lipid membranes during phase transitions

Changes in the ionic permeability of bilayer lipid membranes from dipalmitoyl phosphatidylcholine at temperatures of phase transition in LiCl (1 M) solution after the addition of polyethyleneglycols of different molecular masses have been studied. The transition of ionic membrane channels from the conducting state to a blocking nonconducting state using polymers makes it possible to calibrate lipid pores. It was shown that low-molecular-weight glycerol, polyethyleneglycols with molecular masses of 300 and 600 decrease the amplitude of fluctuations of the current through the membrane, the decrease being proportional to the size of the polymer molecule being incorporated. The addition of polyethyleneglycols with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. This result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as polyethyleneglycols with molecular masses of 6000 and 20000, which are practically not incorporated into the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of polyethyleneglycol. It is assumed that the complete blockade of the conductivity of lipid ionic channels by polyethyleneglycols with molecular masses of 1450, 2000, and 3350 is due to the dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic pore.     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from ?40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.     

On kinetics of phase transitions in cell membranes

Phase transitions in a bicomponent lipid membrane are considered. It is shown that in this case metastable states practically do not arise and phase transitions are smooth and hysteresisless. An elastic frame on the surface of the membrane changes the character of phase transitions: they become sharp and hysteretic. The role of membrane phase transitions for regulation of cell processes is considered.     

The influence of water on the phase transition of sheep lung surfactant. A possible mechanism for surfactant phase transitions in vivo

The effect of water on the thermal properties of sheep lung surfactant lipids was determined by differential scanning calorimetry. Dry surfactant exhibited a phase transition with an upper limit of about 54 degrees C, whereas that of the fully hydrated surfactant was about 30 degrees C. The effect of water was confined to a range of hydration values from 0 to 25%. The results indicate that pulmonary surfactant lipids are capable of undergoing both thermotropic and lyotropic mesomorphism in vitro. The degree of hydration of the surfactant could influence its in vivo biophysical role in alveolar dynamics. Indeed, small changes in the surfactant to water ratio induced by regional differences in the surfactant concentration at the alveolar surface during alveolar expansion and contraction could be sufficient to trigger isothermal phase transitions in the surfactant lipids. This would allow changes to occur in the equilibrium between solidus and fluidus surfactant during the respiratory cycle.     

A model for phase transitions in lipid bilayers and biological membranes

We wish to present an order-disorder model for the observed phase transitions in lipid bilayers and biological membranes. We show that the model may, under certain circumstances, exhibit two phase transitions, one corresponding to positional disordering of entire lipid molecules, and the other corresponding to orientational disordering in the hydrocarbon chains. We then give results of our numerical analysis of the model and compare them with experimental data. Shortcomings of the model and future directions for analyses of this type are also discussed.     

Rupture and transformation of lipid bilayer membranes at thermal phase transitions

Nuclear magnetic resonance was used to study dimyristoylphosphatidylcholine vesicles. Loss of vesicle contents and transformation to more extended bilayer structures near the gel to liquid crystalline phase transition is related to potential cell membrane damage on lowering environmental temperatures.     

Initiation of anion-selective ionic channels in bilayer membranes at lipid phase transitions

A study was carried out to electric parameters of single ionic channels initiated at phase transition of bromidmetilate 1,2-distearoyl-rac-glycero-3-(O-beta-dimethylaminoethyl)-methylphosphonate, whose molecules under conditions given below are possibly charged. It has been shown that changes of transmembrane current appear at phase transition temperature. Comparison between ionic selectivity of channels initiated at Tph.t in the membranes of DSL and its phosphate analog suggests that the channel walls initiated at phospholipid phase transitions are covered with polar groups of molecules.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

Energetics and self-assembly of amphipathic peptide pores in lipid membranes

We present a theoretical study of the energetics, equilibrium size, and size distribution of membrane pores composed of electrically charged amphipathic peptides. The peptides are modeled as cylinders (mimicking alpha-helices) carrying different amounts of charge, with the charge being uniformly distributed over a hydrophilic face, defined by the angle subtended by polar amino acid residues. The free energy of a pore of a given radius, R, and a given number of peptides, s, is expressed as a sum of the peptides' electrostatic charging energy (calculated using Poisson-Boltzmann theory), and the lipid-perturbation energy associated with the formation of a membrane rim (which we model as being semitoroidal) in the gap between neighboring peptides. A simple phenomenological model is used to calculate the membrane perturbation energy. The balance between the opposing forces (namely, the radial free energy derivatives) associated with the electrostatic free energy that favors large R, and the membrane perturbation term that favors small R, dictates the equilibrium properties of the pore. Systematic calculations are reported for circular pores composed of various numbers of peptides, carrying different amounts of charge (1-6 elementary, positive charges) and characterized by different polar angles. We find that the optimal R's, for all (except, possibly, very weakly) charged peptides conform to the "toroidal" pore model, whereby a membrane rim larger than approximately 1 nm intervenes between neighboring peptides. Only weakly charged peptides are likely to form "barrel-stave" pores where the peptides essentially touch one another. Treating pore formation as a two-dimensional self-assembly phenomenon, a simple statistical thermodynamic model is formulated and used to calculate pore size distributions. We find that the average pore size and size polydispersity increase with peptide charge and with the amphipathic polar angle. We also argue that the transition of peptides from the adsorbed to the inserted (membrane pore) state is cooperative and thus occurs rather abruptly upon a change in ambient conditions.     

Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes

The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (<200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.     

Mechanistic interpretation of the influence of lipid phase transitions on transport functions.

In an attempt to understand the mechanism by which a structural change of membrane lipids affects transport functions, the temperature dependence of transport rate has been measured to below the low temperature end of the fluid in equilibrium ordered phase transition of the membrane lipids. The unsaturated fatty acid requiring Escherichia coli strain T105 was supplemented with either trans-delta9-octadecenoate or trans-delta9-hexadecenoate or supplemented with and subsequently starved for cis-delta9-octadecenoate. Fluid in equilibrium ordered phase transitions measured in whole cells using the fluorescence probe N-phenyl-1-naphthylamine were compared with the temperature dependence of beta-glucoside and beta-galactoside transport. In addition to the previously observed downward "break" in the Arrhenius plot of transport rate which occurred near the middle of the phase transition temperature range, a second upward "break" was observed which could be correlated with the low-temperature end of the phase transition. These experiments are interpreted in terms of a partitioning of transport proteins between ordered and fluid domains which is described by a lateral distribution coefficient, k. This distribution coefficient varies with the membrane lipid composition as well as with the transport system. Values for k suggest a 2-20-fold preference for the partitioning of transport proteins into the fluid parts of the membrane.     

Low levels of irradiation modify lipid domains in model membranes: a laser Raman study

We have used Raman spectroscopy to study the effects of ionizing radiation on thermal transitions of dipalmitoyl lecithin + polyunsaturated fatty acid liposomes. Raman spectra in the CH (2800-3000 cm-1), C = C (1600-1680 cm-1), and C-C (1000-1150 cm-1) stretching regions are sensitive to ionizing radiation. The CH stretching of acyl chains yields three strong bands around 2850, 2880, and 2930 cm-1. The ratios of the relative intensities of 2880 and 2850 cm-1 bands, i.e., I2880/2850, when plotted against temperature show multiple infection points which correspond to multiple spectroscopic transitions. These are ascribed to a separate phase with distinctive proportions of lecithin and polyunsaturated fatty acids. We find these transitions sensitive to low levels of ionizing radiation. Doses as low as 5-15 rad after 48 h of 60Co gamma irradiation and 60 kVp X irradiation drastically broaden and shift the polyunsaturated rich phase which occurs at lower temperatures (-7 to +5 degrees C) than that of pure dipalmitoyl lecithin (39 degrees C). In addition a new transition around 46 degrees C also emerges upon irradiation (48 h postirradiation). These irradiation effects can be accelerated by the presence of catalytic amounts of Fe2+/EDTA +H2O2. The membrane transition modification is more sensitive to 60 kVp X rays in comparison to 60Co gamma rays owing to the high LET component of the former. The intensity of 1660 cm-1 band, assigned to C = C stretching in the cis-configuration, loses intensity upon irradiation. Concomitantly, a new band around 1675 cm-1, assigned to trans-configuration, emerges. Similarly the increase in the "order parameter" as calculated from the relative intensities of C--C stretching bands indicates rigidification of membrane. Various factors such as reduction in unsaturation, increase in trans-configuration, and the formation of multiple peroxidation products are invoked as lipid phase modifiers.