Effects of divalent cations,temperature, osmotic pressure gradient,and vesicle curvature on phosphatidylserine vesicle fusion

Summary Fusion of phosphatidylserine vesicles induced by divalent cations, temperature and osmotic pressure gradients across the membrane was studied with respect to variations in vesicle size. Vesicle fusion was followed by two different methods: 1) the Tb/DPA fusion assay, whereby the fluorescent intensity upon mixing of the internal aqueous contents of fused lipid vesicles was monitored, and 2) measurement of the changes in turbidity of the vesicle suspension due to vesicle fusion. It was found that the threshold concentration of divalent cations necessary to induce vesicle fusion depended on the size of vesicles; as the diameter of the vesicle increased, the threshold value increased and the extent of fusion became less. For the osmotic pressure-induced vesicle fusion, the larger the diameter of vesicles, the smaller was the osmotic pressure gradient required to induce membrane fusion. Divalent cations, temperature increase and vesicle membrane expansion by osmotic pressure gradient all resulted in increase in surface energy (tension) of the membrane. The degree of membrane fusion correlated with the corresponding surface energy changes of vesicle membranes due to the above fusion-inducing agents. The increase in surface energy of 9.5 dyn/cm from the reference state corresponded to the threshold point of phosphatidylserine membrane fusion. An attempt was made to explain the factors influencing fusion phenomena on the basis of a single unifying theory.     

Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles.

Aggregation and fusion of unilamellar vesicles consisting of N-acyl-N-methylphosphatidylethanolamine were studied as a function of mono- and divalent cation concentrations. The aggregation reactions were irreversible processes, as demonstrated by changes in monovalent ion concentrations and by the addition of ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations, suggesting the possibility of some cation-induced vesicle fusion. An increase in the NaCl ionic strength of the vesicle suspension solutions diminishes the threshold concentration for Li+ and K+ and increases that corresponding to Mn2+, Mg2+ and Ca2+. However NaCl concentrations above 300 mM yield smaller threshold values for the divalent cation-induced processes, probably due to the increased size of phospholipid vesicles as the ionic strength of the medium increases.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.     

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.     

Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels

A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.     

Fusion,Leakage and Surface Hydrophobicity of Vesicles Containing Phosphoinositides: Influence of Steric and Electrostatic Effects

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the lipid mixing assay. This correlation held even at physiologically relevant small concentrations of PPI in vesicle membranes. However, the presence of nonphosphorylated PI inhibited fusion due to the steric effect of the inositol ring. The cation threshold concentration for the lipid mixing of vesicles made of mixtures of phosphatidylserine (PS) with PI increased with increasing PI contents. For all vesicle systems studied, a decrease in vesicle surface dielectric constant and an increase in vesicle leakage accompanied fusion. The presence of the nonphosphorylated inositol ring in PI did not interfere with the changes in the surface dielectric constant caused by fusogenic cations. Therefore, we deduce that the reduction of the surface dielectric constant is a necessary condition for membrane fusion to occur but it does not correlate with membrane fusion when interacting membranes are blocked for close approach as by the nonphosphorylated inositol ring.     

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.     

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle-membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg²⁺, Ca²⁺, Mn²⁺, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time.When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg²⁺, 7 mM for Sr²⁺, 6 mM for Ca²⁺, 3.5 mM for Ba²⁺ and 1.8 mM for Mn²⁺. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn²⁺ > Ca²⁺ > Mg²⁺ and their critical concentrations were in between the former two cases. The threshold concentrations also depended upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine (1 : 1) vesicles, above the critical concentrations of Mn²⁺ and Ca²⁺, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h.This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Calcium-dependent binding of phosphorylated human pre interleukin 1 alpha to phospholipids

The effect of phosphorylation of pre interleukin 1 alpha (IL 1 alpha) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1 alpha (residues 64 to 271) and phosphorylated this pre IL 1 alpha in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1 alpha selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1 alpha to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100 microM was required. Unphosphorylated pre IL 1 alpha did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1 alpha did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1 alpha bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1 alpha binds to the inner surface of plasma membrane in a Ca2(+)- and phospholipid-dependent manner.     

Studies on the mechanism of membrane fusion: evidence for an intermembrane Ca2+-phospholipid complex, synergism with Mg2+, and inhibition by spectrin.

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents, and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T greater than or equal to 25 degrees C induces the formation of an "anhydrous" complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc greater than 100 degrees C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents, and fusion of the vesicles into larger membranous structures. By contrast, addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T greater than or equal to 20 degrees C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the two cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the erythrocyte protein "spectrin" inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. It is proposed that the unique PS/Ca complex, which involves close apposition of vesicle membranes, is an intermembrane "trans" complex. We further propose that such a complex is a key step for the resultant phase transition and fusion of PS vesicles. By contrast, the PS/Mg complex is proposed to be a "cis" complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.     

Kinetics of a Ca2+-triggered membrane aggregation reaction of phospholipid membranes

Ca²⁺ and other divalent cations can trigger aggregation of phospholipid vesicles containing phosphatidic acid or phosphatidylserine. The reaction, which can be detected by an increase in light scattering, has a critical dependence on the Ca²⁺ concentration, with a threshold near 4 mM Ca²⁺. This is the concentration for half-saturation of the polar head groups and for full neutralization of the membrane surface charge. The aggregation proceeds as a “polymerization” reaction, eventually forming such large aggregates that the vesicles precipitate. The stopped-flow rapid mixing technique was used to study the vesicle dimerization reaction which is the first step in the overall aggregation process. Vesicle dimerization resulted in a doubling of light scattering and had a vesicle concentration-dependent time constant ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) which varied between 0.4 and 2.0 s under the conditions of the study. Analysis of the dependence of the reaction amplitude and $\text{1}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ on the concentrations of vesicles and Ca²⁺ showed that the Ca²⁺ binding is fast, and that the dimerization proceeds by a mechanism in which the vesicles first collide to form an encounter complex followed by a slower conversion of the encounter complex to a stable complex. For phosphatidic acid vesicles, about 200–700 collisions are necessary to achieve a stable dimer. The rate-limiting step in the overall reaction in thus the transformation of the encounter complex into a stable complex, requiring 0.5 and 1.0 ms. The above-mentioned results are relatively insensitive to the type of divalent cation or to the choice of negatively charged lipid (phosphatidic acid or phosphatidylserine).Evidence is given that the stable complex is effected by Ca²⁺-mediated salt bridges between the two membranes and that the rate constant of the transformation step derives from the statistics of the distribution and the rate of redistribution of Ca²⁺-occupied polar head groups on the membrane surfaces. The relevance of these results to the problem of Ca²⁺-induced fusion of biological membranes is discussed.     

The fusogenic effect of synthetic polycations on negatively charged lipid bilayers

The effect of synthetic polycations, polyallylamine, and polyethylenimine, on liposomes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1:4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05 mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multicycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, polyallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phosphatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidylserine vesicles at about 1 mM at the liposomal concentrations investigated.     

Divalent cation transport by vesicular nucleotide transporter

The vesicular nucleotide transporter (VNUT) is a secretory vesicle protein that is responsible for the vesicular storage and subsequent exocytosis of ATP (Sawada, K., Echigo, N., Juge, N., Miyaji, T., Otsuka, M., Omote, H., and Moriyama, Y. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 5683-5686). Because VNUT actively transports ATP in a membrane potential (Δψ)-dependent manner irrespective of divalent cations such as Mg(2+) and Ca(2+), VNUT recognizes free ATP as a transport substrate. However, whether or not VNUT transports chelating complexes with divalent cations remains unknown. Here, we show that proteoliposomes containing purified VNUT actively took up Mg(2+) when ATP was present, as detected by atomic absorption spectroscopy. The VNUT-containing proteoliposomes also took up radioactive Ca(2+) upon imposing Δψ (positive-inside) but not ΔpH. The Δψ-driven Ca(2+) uptake required ATP and a millimolar concentration of Cl(-), which was inhibited by Evans blue, a specific inhibitor of SLC17-type transporters. VNUT in which Arg-119 was specifically mutated to alanine, the counterpart of the essential amino acid residue of the SLC17 family, lost the ability to take up both ATP and Ca(2+). Ca(2+) uptake was also inhibited in the presence of various divalent cations such as Mg(2+). Kinetic analysis indicated that Ca(2+) or Mg(2+) did not affect the apparent affinity for ATP. RNAi of the VNUT gene in PC12 cells decreased the vesicular Mg(2+) concentration to 67.7%. These results indicate that VNUT transports both nucleotides and divalent cations probably as chelating complexes and suggest that VNUT functions as a divalent cation importer in secretory vesicles under physiological conditions.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.     

Phosphatidate and oxidized fatty acids are calcium ionophores. Studies employing arsenazo III in liposomes

Liposomes which have entrapped the metallochromic dye, arsenazo III, constitute a sensitive assay system for ionophoresis of divalent cations. By this means we have compared known calcium ionophores (A23187, ionomycin) with membrane phospholipids, fatty acids, prostanoids, and retinoids. Added at micromolar concentrations to preformed multilamellar liposomes (phosphatidylcholine 7:dicetyl phosphate 2: cholesterol 1) both A23187 and ionomycin, as well as phosphatidic acid and products derived from linoleic acid, linolenic acid, and two eicosatrienoic acids provoked Ca influx (e.g. phosphatidic acid: 0.13 mol of Ca2+/mol of membrane lipid/5 min). A variety of other phospholipids (e.g. phosphatidylinositol), fatty acids (e.g. arachidonic acid), prostanoids (e.g. PGE1) retinoids (e.g. retinoic acid), and glyceryl ether phosphorylcholines ("platelet-activating factors") were without effect. Phosphatidic acid and oxidized fatty acids translocated divalent cations selectively, demonstrating the same rank order as A23187 or ionomycin: Mn greater than Ca greater than Sr much greater than Mg. Membrane lysis did not contribute to the perceived translocation; the liposomes remained impermeable to EDTA, EGTA, arsenazo III, or Mg. Liposomes with phosphatidic acid or oxidized trienoic acids preincorporated at 1-5 mole % of total lipids also permitted translocation of Ca but not Mg. Reduction of ionophoretic fatty acids or ionomycin with stannous chloride abolished their ionophoretic activity. Release of Ca from liposomes which had entrapped arsenazo III-Ca complexes into a medium rich in EGTA permitted calculation of efflux induced by ionophores, whether these were added to the outside of liposomes or preincorporated. Data suggest that phosphatidic acid and oxidized di- and trienoic fatty acids, which act as calcium ionophores in model bilayers, could serve as "endogenous ionophores" in cells.     

Fusion of phosphatidylserine and mixed phosphatidylserine-phosphatidylcholine vesicles. Dependence on calcium concentration and temperature

Dynamic light scattering has been used to study the temperature dependence of Ca2+-induced fusion of phosphatidylserine vesicles and mixed vesicles containing phosphatidylserine and different phosphatidylcholines. The final vesicle size after Ca2+ and EDTA incubation serves as a measure of the extent of fusion. With phosphatidylserine vesicles, the extent of fusion shows a sharp maximum at an incubation temperature which depends on the Ca2+ concentration between 0.8 and 2 mM. The shift in the fusion peak temperature with Ca2+ concentration is similar to the typical shift in the phase transition temperature with divalent cation concentration in acidic phospholipids. The results suggest a direct correlation between the fusion peak temperature and the phase transition temperature in the presence of Ca2+ prior to fusion. With mixed vesicles containing up to 33% of a phosphatidylcholine in at least 2 mM Ca2+, the extent of fusion as a function of incubation temperature also shows a maximum. The fusion peak temperature is essentially independent of the quantity and type of phosphatidylcholine and the Ca2+ concentration, and identical to that with pure phosphatidylserine in excess Ca2+. The results imply that Ca2+- induced molecular segregation occurs first, and fusion subsequently takes place between pure phosphatidylserine domains.     

Binding of monovalent cations to phosphatidylserine and modulation of Ca2+- and Mg2+-induced vesicle fusion

The effect of several monovalent cations on the Ca2+-induced aggregation and fusion of sonicated phosphatidylserine (PS) vesicles is studied by monitoring the mixing of internal compartments of the fusing vesicles using the Tb/dipicolinic acid assay. The dissociation of the fluorescent Tb-dipicolinate complex which accompanies Ca2+-induced vesicle fusion is determined directly and is due to leakage of contents and entry of medium into vesicles. PS vesicles do not fuse when the medium contains only monovalent cations (at pH 7.4), regardless of the cation concentration or whether there is aggregation of the vesicles. A mass-action kinetic analysis of the data provides estimates for the rate of aggregation, C11, and for the rate of fusion per se, f11. Values of f11 increase dramatically with reduction in monovalent cation concentration and are primarily determined by binding ratios of Ca2+ or Mg2+ per PS. With 300 mM of monovalent cations, the fusion per se is essentially rate-limiting to the overall fusion process and values of f11 are significantly larger with the monovalent cations which bind the least, i.e., according to the sequence tetramethylammonium greater than K+ greater than Na+ greater than Li+. With monovalent cations in concentrations of 100 mM or less, the aggregation is rate-limiting to the fusion and the overall initial fusion rates are determined by an interplay between aggregation and fusion rates. Under conditions of fast aggregation, the Ca2+-induced fusion of small PS vesicles can occur within milliseconds or less.