Phosphatidylserine Membranes Research Articles

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.

Characterization of the channel properties of tetanus toxin in planar lipid bilayers

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.

Fluorescent probes of electrostatic potential 1 nm from the membrane surface.

We measured the electrostatic potential 1 nm from the surface of charged phospholipid bilayer membranes to test the predictions of the Gouy-Chapman theory. Fluorescent probes (anthraniloyl, 5-(dimethylamino)naphthalene-1-sulfonyl, Lucifer yellow) were attached covalently to the sialic acid residue of the ganglioside galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylc eramide (GM1). These fluorescent gangliosides were incorporated into neutral [phosphatidylcholine (PC)] or charged [phosphatidylserine (PS)] phospholipid bilayers, and the fluorescence was quenched with the cations thallium and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (tempamine). We calculated the electrostatic potential at the chromophore from the quenching ratio using the Boltzmann relation: the average potential was -30 mV for PS bilayers in 0.1 M NaNO3. We assume the chromophore is 1 nm from the surface because X-ray diffraction measurements demonstrate that the sialic acid residue of GM1 is 1 nm from the surface of a PC/GM1 bilayer [McDaniel, R. V., & McIntosh, T. J. (1986) Biophys. J. 49, 94-96]. We also used thallium and tempamine to quench the fluorescence of chromophores located at the surface of the PS membranes; in 0.1 M NaNO3 the average surface potential was -80 mV, which agrees with other measurements. The Gouy-Chapman theory predicts that the potential 1 nm from a membrane with a surface potential of -80 mV is -24 mV; this prediction agrees qualitatively with the experimental results obtained with fluorescent gangliosides.

Effects of calcium on the gramicidin A single channel in phosphatidylserine membranes

In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979). The aim of this paper is to make a quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 mM KCl in the presence of different amounts of CaCl2, MgCl2 or TEACl. The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K+ activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions.

Effects of cations and polyamines on the aggregation and fusion of phosphatidylserine membranes.

Effects of various metal cations and polyamines on aggregation and fusion of phosphatidylserine vesicles and their associated physicochemical properties (such as surface tension and vesicle electrophoretic mobility) have been studied. It was found that metal polycations and hydrogen ion caused an increase in the surface tension of a phosphatidylserine monolayer, whereas the polyamines and other monovalent cations did not increase the surface tension of the membrane appreciably. All cations used affected the vesicle mobility roughly in the order of the number of their valencies and linearly with respect to the logarithm of their concentrations of ions; vesicle surface charge densities are reduced by adsorption and screening of the counter ions depending on their valencies and concentrations. The degree of aggregation of lipid vesicles parallels somewhat that of the reduction of vesicle electrophoretic mobilities. However, the degree of membrane fusion induced by these cations parallels that of the increase in surface tension of the membranes induced by these cations.

Dynamic approach to self-organization or a phase transition. Model for a non-equilibrium phase transition in phosphatidylserine membranes by the binding of Ca 2+ and Cu 2+

A self-organized chemical system is described by a set of rate equations for a primary and a partial system. The partial system acts as an internal driving force to regulate the primary system. Thermal equilibrium of the primary system is broken by coupling with the partial system and there is a change into a new state through quasi-equilibrium. The instability of the quasi-equilibrium is treated by a transition parameter k' 3 describing an autocatalytic reaction. For k' 3 > 0 the state is stable, while for k' 3 < 0 it is transferred into a new state. We treat a phase transition of head groups of phosphatidylserine molecules in membranes by the binding of Ca 2+ and Cu 2+ using this model.

Effects of calcium on permethrin-phosphatidylserine interactions in model membranes

The effects of permethrin on lipid packing order in liposomal membranes containing phosphatidylserine (PS) have been examined in the presence and absence of calcium. Steady-state fluorescence anisotropy and phase-modulation techniques were utilized with the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH), trans-parinaric acid, and cis-parinaric acid to determine alterations in lipid packing by permethrin as a function of temperature. In general, permethrin exhibited disordering effects only in gel phase lipid. In pure PS membranes, permethrin had little effect on small unilamellar vesicles (SUVs). However, substantial reductions in lipid packing order were detected by decreases in DPH fluorescence anisotropy when these SUVs underwent isothermal fluid to gel phase transition upon exposure to Ca 2+. Although Ca 2+ increased the main PS phase transition temperature ( T c ), a small residual phase transition was detectable by parinaric acid anisotropy and lifetimes within the temperature range corresponding to the SUV phase transition. Permethrin smoothed this residual transition, suggesting that this compound altered a process which was preventing a portion of PS from undergoing the Ca 2+-mediated isothermal transition to the solid phase. In mixed liposomes containing dipalmitoyl phosphatidylcholine (DPPC) at a PS:DPPC ratio of 3:2, the T c of the SUVs was increased while that of the Ca 2+-treated vesicles was decreased. As in the pure PS system, permethrin had a greater disordering effect upon the lipid packing in the Ca 2+-treated membranes. There was a slight disordering effect by permethrin in PS:DPPC SUVs at temperatures below the T c , indicating the existence of DPPC enriched domains in the gel phase lipid which were sensitive to permethrin. The results demonstrate that Ca 2+-mediated changes in membrane characteristics, such as isothermal fluid to gel phase transition, can influence membrane sensitivity to the lipid disordering effects of permethrin.

Effects of cations and polyamines on the aggregation and fusion of phosphatidylserine membranes

Effects of various metal cations and polyamines on aggregation and fusion of phosphatidylserine vesicles and their associated physicochemical properties (such as surface tension and vesicle electrophoretic mobility) have been studied. It was found that metal polycations and hydrogen ion caused an increase in the surface tension of a phosphatidylserine monolayer, whereas the polyamines and other monovalent cations did not increase the surface tension of the membrane appreciably. All cations used affected the vesicle mobility roughly in the order of the number of their valencies and linearly with respect to the logarithm of their concentrations of ions; vesicle surface charge densities are reduced by adsorption and screening of the counter ions depending on their valencies and concentrations. The degree of aggregation of lipid vesicles parallels somewhat that of the reduction of vesicle electrophoretic mobilities. However, the degree of membrane fusion induced by these cations parallels that of the increase in surface tension of the membranes induced by these cations.

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers.

A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K+ concentration that could not be described by simple Michaelis-Menten saturation. At low K+ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4 m KCl. At the same Ca2+ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K+ and Ca2+ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K+-conduction and Ca2+-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 A from the surface.

Divalent cation-induced phosphatidic acid membrane fusion. Effect of ion binding and membrane surface tension

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb 3+ /dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the ‘threshold’ concentration to induce massive vesicle membrane fusion was the same for Ca 2+ and Mg 2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn / cm for both Ca 2+ and Mg 2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.

Steady state photocurrents associated with electron transfer through planar lipid bilayers sensitized by zinc 5,10,15,20-tetraphenylporphyrins

Bilayer phosphatidylserine membranes sensitized by zinc tetraphenylporphyrin (ZnTPP) and its 1-(CH 2) 2 CO 2H derivative (ZnTPP-Ac) are formed between one oxidant (Fe 3+) and one reductant (ascorbic acid) solution. Under visible light illumination steady state photocurrents are recorded. The acidic derivative, probably due to its better interfacial location, is 30 times as efficient as ZnTPP. Linear relationships are observed between the inverse of the stationary photocurrent and the following functions: 1/[A]; 1/ E and exp (−0.3 fU); [A] being the electron acceptor concentration; E, the light intensity and U, the external applied voltage. A model based on a translocation of the oxidized ZnTPP-Ac accounts for these experimental relationships.

Synergetic effects of Ca 2+ and Cu 2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant ( K H 2O ) of the coordinated H 2O, such as Ca 2+ and Cu 2+, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by 1H-NMR and ESR using Ca 2+ and / or Cu 2+, which has a similar K H 2O to that of Ca 2+. We found that Ca 2+ did not hinder the molecular movements of PS. However, Cu 2+ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca 2+ and Cu 2+, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.

Phosphorus-31 and carbon-13 nuclear magnetic resonance studies of divalent cation binding to phosphatidylserine membranes: use of cobalt as a paramagnetic probe.

The paramagnetic divalent cation cobalt has large and well-understood effects on NMR signals from ligands bound in the first coordination sphere, i.e., inner-sphere ligands, and we have used these effects to identify divalent cation binding sites at the surface of phosphatidylserine membranes. 31P NMR results show that 13% of the bound cobalt ions are involved in inner-sphere complexes with the phosphodiester group, while 13C NMR results show that 54% of the bound cobalt ions are involved in unidentate inner sphere complexes with the carboxyl group. No evidence is found for cobalt binding to the carbonyl groups, but proton release studies suggest that 32% of the bound cobalt ions are involved in chelate complexes that contain both the carboxyl and the amine groups. All (i.e., 13% + 54% + 32% = 99%) of the bound cobalt ions can thus be accounted for in terms of inner sphere complexes with the phosphodiester group or the carboxyl group. We suggest that the unidentate inner-sphere complex between cobalt and the carboxyl group of phosphatidylserine and the inner-sphere complex between cobalt and the phosphodiester group of phosphatidylserine provide reasonable models for complexes between alkaline earth cations and phosphatidylserine membranes.

A mechanism of divalent ion-induced phosphatidylserine membrane fusion

A mechanism for the divalent cation-induced membrane fusion of phosphatidylserine membranes is proposed. Fusion was followed by the Tb/DPA (dipicolinic acid) assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of unilamellar lipid vesicles, and the threshold concentrations required for various divalent cations to induce membrane fusion were determined from the fluorescence spectrum of the lipid vesicle suspension with respect to various concentrations of divalent ions. Also, the surface tension of monolayers made of the same lipids as used in the fusion experiments was measured with respect to the variation of divalent cation concentrations. The surface tension increase in the monolayer, induced by changing divalent ion concentrations from zero to a concentration which corresponded to its threshold concentration to induce vesicle membrane fusion, was the same (approx. 8 dyn/cm) for all divalent ions used. From these experimental data and the theory concerning ion binding to the membrane, it is deduced that the main cause of divalent cation-induced membrane fusion of phosphatidylserine membranes is the degree of increased hydrophobicity (surface tension increase) of the membrane surface, which results from the binding of cations to acidic phospholipid membrane surfaces. Some discussion on the molecular mechanism of phospholipid membrane fusion is given.

Two purified fractions of alamethicin have different conductance properties

The properties of two purified alamethicin fractions, Fraction 4 and Fraction 6, have been studied in phosphatidylethanolamine (PE) membranes and phosphatidylserine (PS) membranes. Membranes doped with Fraction 4 show well-defined single channel conductance (mean lifetime about 20 ms). The autocorrelation function of the current fluctuations has one relaxation time of the same order as the mean lifetime of the single channels, and the current response to a voltage pulse follows an exponential with only one time constant. The conductance of a membrane doped with Fraction 6 has a voltage-independent part and a current-voltage curve with a slope that is half the slope of the Fraction 4 current-voltage curve. In the presence of Fraction 6, PS membranes and PE membranes both have symmetrical current-voltage curves even with Fraction 6 added to only one side. We did not detect any well-defined single channel levels in the presence of Fraction 6, and autocorrelation analysis of the current due to Fraction 6 gave two characteristic correlation times: a fast time (about 5 ms) and a slow time (about 50 ms). High current level kinetics of Fraction 6 also show two time constants. A possible explanation for the differences between the two fractions is that Fraction 6 monomers have a lower dipole moment than those of Fraction 4. The difference in channel stability can be explained by a lowered tendency of the monomers to line up parallel to the field. The negative branch and voltage-independent conductance can be explained by lowered energy of insertion of monomers into the membrane, and lowered energy of interaction between the monomers and the electric field.

Surface potential of phosphatidylserine monolayers II. Divalent and monovalent ion binding

Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface potential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various monovalent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca 2+, Mg 2+, Na + and (Me) 4N + binding constant values of 30 M −1, 10 M −1, 0.6 M −1 and 0.05 M −1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H + in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H + for biomolecules was made in terms of ion binding due to other ions at the same site as for H + in biomolecules.

The exothermic reaction of calcium with unilamellar phosphatidylserine vesicles: Titration microcalorimetry

Phospholipid membranes have been used extensively as model systems for understanding the mechanism of membrane fusion in biological systems (reviews [I ,2]). Phosphatidylserine (PS) membranes have unusual properties with respect to their interaction with divalent ions. For example, planar bilayer membranes (black lipid films) are unstable if Ca*+ is added to only one side of the membrane [3,4]. Small unilamellar PS vesicles have a similar instability in the presence of Ca*+ in that they fuse [5], leak their contents [3], and eventually form cochlear cylinders [6]. PS also exhibits a specificity for the binding of Ca*+, and the gel to liquid crystalline transition temperature of the phospholipid is shifted to above 100°C when Ca*’ is added [7,8]. The binding of divalent ions to PS appears to be correlated with their ability to fuse sonicated vesicles made of this phospholipid [8-l I]. The addition of Ca*+ to these vesicles results in the release of heat as measured in a batch calorimeter [8]. To identify the source of the heat released and its possible involvement in the fusion reaction requires a detailed knowledge of the relationship of the extent of the exothermic reaction and the amount of Ca*+ bound to the vesicles. For this purpose, we have measured the heat of reaction in a titrating microcalorimeter and have determined the Ca*+ bound by means of a

Capacitance and conductance as tools for the measurement of asymmetric surface potentials and energy barriers of lipid bilayer membranes

A simple method for determination of asymmetric surface potentials in lipid bilayers is described. The method is based on the dependence of bilayer capacitance on transmembrane voltage. The capacitance is measured by rectifying in 90 degrees component of an applied alternating current signal. A superimposed slow triangular wave results in a hysteresis-like time course of capacitance. The center of the hysteresis figure is shifted along the voltage axis by an amount equal to the difference of the dipole plus surface-charge potentials on the two sides of the bilayer (capacitance minimization potential). Alternatively, such bilayer asymmetry was studied by using the current-voltage characteristics in the presence of nonactin as a carrier. This analysis was based on the integrated Nernst-Planck equation, assuming a trapezoidal energy barrier and equilibrium of the surface reactions. The two methods gave consistent results for the surface potentials of phosphatidyl serine membranes asymmetrically shielded with calcium. In addition, the current analysis yields the positions of the corners of the barrier, found to be set in 13% for this lipid.

Effects of surface charge on the conductance of the gramicidin channel

The electric conductance of the cation-permeable gramicidin channel in negatively charged phosphatidylserine membranes has been studied. At low electrolyte concentrations the single-channel conductance is much larger in the negatively charged membrane than in a neutral membrane. This enhancement of conductance is in agreement with theoretical expectations, although a complete description of the salt concentration dependence of conductance was not possible. The results of these experiments may be compared with previous studies of a negatively charged gramicidin analog ( O-pyromellityl gramicidin). It is found that the electrostatic effect on the conductance is much larger for a neutral channel embedded in a negatively charged lipid than for the negatively charged O-pyromellityl analog (with three charges at the channel mouth) embedded in a neutral lipid.

Variability of conductivity changes in black phosphatidylserine membranes induced by proteins from erythrocyte membranes.

The electrical conductivity of black phosphatidylserine membranes, in solutions of 100 mM NaCl, 10 mM sodium phosphate (pH 7.1), is strongly increased by the intrinsic proteins ("strongly bound" protein fraction) from human erythrocyte membranes. The magnitude of the conductivity increase is highly dependent on the maximum pH-value pH used during the preparation of the protein (8.0 less than or equal to pH less than or equal 11.8). For each pH, membrane conductivity lambdaf and protein concentration c are linked by the equation lambdaf=k-cs, k and s being functions only of pH. The value of s varies between 1.0 (pH 8) and 4.0 (pH 10). It is assumed that the protein-induced conducting sites, at least for protein pretreatment at pH less than or equal to 10, are assembled from four protein subunits. The incorporation of the subunits into the lipid bilayer is supposed to occur either as the final tetramer (pH 8) or as monomers (pH 10) and possibly dimers (pH around 9).