Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.     

The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.     

Influenza virus hemagglutinin-induced cell-planar bilayer fusion: quantitative dissection of fusion pore kinetics into stages

Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quiescent perfusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops into a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) with a large conductance on the order of one microSiemens. We have studied how virus strain, HA receptors in the target bilayer membrane, and cytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, waiting times were very sensitive to the virus strain and presence of gangliosides (HA receptors) in the bilayer. When bilayers contained gangliosides, the waiting times in the Q stage for cells infected with the PR/8/34 strain of virus were exponentially distributed, whereas waiting times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for PR/8/34 infected cells was complex. The waiting times in the R and S stages of pore growth were exponentially distributed under all tested conditions. Within the R stage, we analyzed the kinetics of the flickering pore by fitting the open and closed time distributions with a sum of two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affected by virus strain or gangliosides. Colchicine and cytochalasin B increased the flicker rates, without affecting the waiting time in the R stage. We conclude that variations in amino acid sequences of the HAs and the presence of gangliosides as receptors within the target membrane critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.     

Gangliosides Affect Membrane-Channel Activities Dependent on Ambient Temperature

1. The functional properties of biological membranes depend on their molecular composition. In regard to this, charged glycosphingolipids play an outstanding role in the functional adaptation of membranes to different temperatures.2. In order to shed some light on the respective functional properties of complex membraneous glycosphingolipids, the effects of altered temperatures (5–40°C) on planar lipid bilayers made from diphytanoylphosphatidylcholine (DPPC) and alamethicin as an ion channel was analyzed in the presence of either a sialoglycosphingolipid (less polar disialoganglioside GD1a or highly polar tetrasialoganglioside GQ1b) or phosphatidylserine (PS; as control).3. Different to the control bilayers made from DPPC or DPPC + PS, the bilayers containing gangliosides had specific maxima in alamethicin conductance and stabile life times. Changes in pore-state conductances indicate structural effects based on an interaction of the large (negatively charged) ganglioside headgroups with the alamethicin pores.4. The results concerning open time and closed time of channels seem to be based on the gangliosides changing the viscosity of the bilayer and possibly introducing phase transitions.5. Thus, the findings suggest that gangliosides (1) directly affect channel molecules via their headgroups and (2) may additionally affect the fluidity of membranes in order to maintain membrane homeoviscosity in areas surrounding ion channels independent from the environmental temperature.6. The effects of gangliosides may be of special interest in describing the ability of neuronal adaptation of vertebrates to temperature and more general regarding the functional adaptation of neurons.     

An amphipathic peptide from the C-terminal region of the human immunodeficiency virus envelope glycoprotein causes pore formation in membranes.

The peptide fragment of the carboxy-terminal region of the human immunodeficiency virus (HIV) transmembrane protein (gp41) has been implicated in T-cell death. This positively charged, amphipathic helix (amino acids 828 to 848) of the envelope protein is located within virions or cytoplasm. We studied the interaction of the isolated, synthetic amphipathic helix of gp41 with planar phospholipid bilayer membranes and with Sf9 cells using voltage clamp, potentiodynamic, and single-cell recording techniques. We found that the peptide binds strongly to planar membranes, especially to the negatively charged phosphatidylserine bilayer. In the presence of micromolar concentrations of peptide sufficient to make its surface densities comparable with those of envelope glycoprotein molecules in HIV virions, an increase in bilayer conductance and a decrease in bilayer stability were observed, showing pore formation in the planar lipid bilayers. These pores were permeable to both monovalent and divalent cations, as well as to chloride. The exposure of the inner leaflet of cell membranes to even 25 nM peptide increased membrane conductance. We suggest that the carboxy-terminal fragment of the HIV type 1 envelope protein may interact with the cell membrane of infected T cells to create lipidic pores which increase membrane permeability, leading to sodium and calcium flux into cells, osmotic swelling, and T-cell necrosis or apoptosis.     

Permeability and electrical properties of planar lipid membranes from thylakoid lipids.

Electrical measurements were carried out on planar lipid membranes from thylakoid lipids. The specific capacitance of membranes formed from decane-containing monogalactosyldiacylglycerol (MGDG), which accounts for 57% of the total lipid content of thylakoids, showed that it adopted a bilayer structure. Solvent-free bilayers of MGDG were not formed, with very rare exceptions, indicating that decane is required to stabilize the planar conformation. However, this cone-shaped lipid produces bilayer structures in combination with other cylindrical thylakoid lipids even in the absence of organic solvent. We compared the properties of solvent-free and decane-containing bilayers from MGDG, soybean lecithin, and the quaternary mixture of lipids similar to that found in vivo. The conductance of decane-MGDG was 26 times higher than that of decane-lecithin. The flux through the decane-lecithin bilayer was found to be slightly dependent on pH, whereas the decane-MGDG membrane was not. The specific conductance of bilayers formed from the quaternary mixture of lipids was 5 to 10 times larger than lecithin (with alkane or not). Further experiments with bilayers made in the presence of a KCl gradient showed that decane-MGDG, decane-MGDG/DGDG/SQDG/PG, and solvent-free MGDG/DGDG/SQDG/PG were cation-selective. The permeability coefficient for potassium ranged from 4.9 to 8.3 x 10(-11) cm s-1. The permeability coefficient for protons in galactolipids, however, was determined to be about six orders of magnitude higher than the value for potassium ions. The HCl permeation mechanism through the lipid membranes was determined from diffusion potentials measured in HCl gradients. Our results suggest that HCl was not transported as neutral molecules. The data is discussed with regard to the function of galactolipids in the ion transport through thylakoid membranes.     

Polymerization in black lipid membranes

A variety of different lipids containing dienoyl groups in the side chains were tested for membrane formation using the planar lipid bilayer approach. One of these lipids formed stable bilayers which could be polymerized using UV-illumination. The influence of the polymerization was studied in monolayers, lipid vesicles and planar bilayers. The stability of the lipid bilayer membranes was increased by polymerization. Thus, the lifetime of the membranes increased from about 1 h to 4–5 h or longer. Furthermore, the specific conductance of unmodified membranes and of carrier-mediated transport is reduced. The transport of lipophilic ions was investigated as a function of polymerization using the charge-pulse method. The absorption of dipicrylamine (DPA^-) is not affected. The translocation of this compound and of tetraphenylborate (B(Ph) ₄ ^- ) showed a strong decrease with polymerization time. The influence of polymerization on the membrane structure may be explained on the basis of a strong viscosity increase in the lipid bilayer membrane.     

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 μm 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.     

Congregation of gangliosides at the junction between two model membranes.

The diversity and distribution of gangliosides in vertebrate tissue suggests an important role in cellular recognition. Two types of experiments are reported to test the hypothesis that gangliosides can congregate to form an adhesive junction between two membranes. First, to monitor ganglioside distribution and mobility in different regions of two large spherical bilayer membranes, fluorescent derivatives of natural gangliosides were synthesized. Second, the cation carrier nonactin was used as a conductance probe to measure the membrane surface potential, which would be altered if there were a redistribution of the charged gangliosides. These studies were conducted in large spherical artificial membranes made from egg phosphatidylcholine or oleoylpalmitoylphosphatidylcholine with 0-12 mol % bovine brain gangliosides dissolved in n-decane. The fluorescent gangliosides utilized were lucifer yellow adducts to the sialic acids (LY-gangliosides) or a cis-paranaric acid substitution of the N-acyl moiety in the ceramide portion of gangliosides GM1 and GD1a (paranaryl-GM1 and paranaryl-GD1a). The polarized fluorescence from the adhesive junction between two membranes containing LY-gangliosides or either paranarylganglioside was compared to that in nonadhesive regions. For LY-gangliosides, total fluorescence in the junction decreased with time, possibly due to electrostatic repulsion of this highly charged derivative. For paranarylgangliosides, fluorescence in the junction increased 7-fold with time, suggesting congregation of this ganglioside. In both cases, a measure of rotational mobility, fluorescence anisotropy, increased dramatically, about 2-fold, as expected for restricted mobility of adhesive compounds. Independent evidence for congregation of charge-bearing gangliosides was found with the conductance probe nonactin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel

Summary A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.The authors would like to thank U. Heber for many helpful discussions. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 176, projects B3 and B7) and by the Fonds der Chemischen Industrie.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa

The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single-channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented.     

Fusion in phospholipid spherical membranes. I. Effect of temperature and lysolecithin.

A study concerning membrane contact and fusion phenomena was made for phospholipid spherical bilayer systems with respect to temperature. Specific temperatures were obtained for the spherical bilayer membranes of phosphatidyl choline (PC) and phosphatidyl serine (PS) which indicated a greater degree of membrane fusion and were designated Tf (the fusion temperature -- PC: 43 degrees C, PS: 38 degrees C). These temperatures were reduced by about 10 degrees C for the membranes incorporated with 20% lysophosphatidyl choline. The results of the contact and fusion observed in the spherical membranes are compared and discussed with the conductance characteristics of the PC and PS planar bilayer membranes as well as dissolution study on the phospholipid monolayers formed at the air/water interface with respect to temperature. Also, a possible molecular mechanism of membrane fusion is discussed in terms of the fluidity and instability of the membrane.     

Evidence that nystatin may not form channels in thin lipid membranes.

The mechanism of nystatin-induced conductance increases in planar bilayer membranes formed from lecithin/cholesterol (1:1) has been examined by spectral analysis of kinetic fluctuations. Lorentzian components of the power spectrum, characteristic of channel activity, were not observed for nystatin-doped membranes. Instead, the noise associated with this system (approximately 10(-27)A2/Hz) was in better agreement with that expected of a carrier mechanism involved in the transport of single charges in uncorrelated fashion. The implications of this result with respect to previous models of nystatin-induced conductance increases are discussed.     

Interaction of furosemide with lipid membranes

Summary The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry,. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.Abbreviations DSC differential scanning calorimetry - PLM planar lipid membranes - DPPC dipalmitoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - PE phosphatidyl ethanol     

Single channel H+ currents through reconstituted chloroplast ATP synthase CF0-CF1.

The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes, blocked the channel almost completely. Our results, which reveal a high channel unit conductance, and at membrane voltages less than 100 mV low open probability with concomitant mean open times in the micros timescale (less than 100 micros) for the energy coupling in the enzyme complex. At physiological membrane voltages for photophosphorylation (about 30 mV) the enzyme complex would then display a time-averaged conductance of about 1 fS.     

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calciuminduced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.