HIV Entry and Envelope Glycoprotein-mediated Fusion

HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process.     

Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109

PDC-109, the major heparin-binding protein of bull seminal plasma, binds specifically to sperm choline lipids at ejaculation and mediates capacitation by stimulating cholesterol and phospholipid efflux. We carried out a biophysical study to investigate the membrane perturbation effect caused by PDC-109. Binding of PDC-109 to phosphatidylcholine model membranes was maximal at a 12:1 phosphatidylcholine to protein molar ratio. The process was independent of the membrane structure and involved a slight conformational change of the protein, compatible with an increased exposure to the solvent. PDC-109 binding to dimyristoylphosphatidylcholine prevented lipid molecules from participating in the gel-to-liquid phase transition, due to enhancement of both acyl chain disorder and interfacial hydration. Visualization of the lipid-protein complexes by electron microscopy showed surface irregularities and the presence of 10-nm particles. Permeability assays confirmed the PDC-109-induced disruption of the vesicles. This effect was not modified by heparin. However, presence of cholesterol inhibited the process in a concentration-dependent manner.     

Lipid-Membrane Affinity of Chimeric Metal-binding Green Fluorescent Protein

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.This revised version was published online in June 2005 with a corrected cover date.     

Exocytotic steps in cell-free system after cholesterol deprivation in synaptosomal plasma membranes and synaptic vesicles

Using a cell-free system we investigated a specific role of cholesterol in exocytotic processes. To modulate the cholesterol content in membrane methyl-beta-cyclodextrin was used as a cholesterol binding agent. The experimental conditions for cholesterol depletion from synaptosomal membrane structures were determined and depended on methyl-beta-cyclodextrin concentration, time and mediums temperature. The role of cholesterol was studied on the stages of synaptic vesicles docking and Ca(2+)-stimulated fusion which are the components of multivesicular compound exocytosis. Using dynamic light scattering technique we have found that after cholesterol depletion from synaptic vesicles the process of their aggregation (docking) remains unchanged. It was found that the rate of calcium-triggered fusion of synaptic vesicles depends on the membrane level of cholesterol. The decreasing level of synaptosomal plasma membrane cholesterol by 8% leads to suppression of the Ca(2+)-dependent membrane fusion with synaptic vesicles. But, under 25% reduction of plasma membrane cholesterol the level of membrane merging with synaptic vesicles did not differ from control; probably this is due to changes in physical properties of lipid bilayer and/ or disturbances in function of membrane proteins driving this process. In cholesterol depleted synaptosomes the exocytotic release of glutamate stimulated by calcium was decreased by 32%. Obtained data suggest that the cholesterol concenration in synaptosomal plasma membranes or synaptic vesicles is the crucial determinant for synaptic transmission efficiency in nerve terminals.     

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30?mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30?mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.     

Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential

Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.     

Modulation of myelin basic protein-induced aggregation and fusion of liposomes by cholesterol, aliphatic aldehydes and alkanes

The effect of cholesterol on myelin basic protein-induced aggregation of zwitterionic phospholipid vesicles was studied by turbidimetry, quasi-elastic light scattering and centrifugation techniques. Without cholesterol, the degree of vesicle aggregation caused by myelin basic protein is relatively low and is only slightly increased using cholesterol concentrations up to approx. 25-30 mol%. When the cholesterol content in the bilayer exceeds approx. 30 mol%, there is a dramatic increase in the susceptibility of the vesicles to aggregation in the presence of myelin basic protein. Palmitoyl aldehyde and eicosane, substances resembling products of lipid degradation, increase myelin basic protein promoted fusion of vesicles. The fusion is accompanied by increased leakage of entrapped carboxyfluorescein. In the presence of cholesterol, myelin basic protein-induced fusion of the liposomes becomes much more sensitive to the presence of aliphatic aldehydes or alkanes. The results suggest that cholesterol has an important role in promoting membrane adhesion in biological systems but these structures become unstable in the presence of small amounts of products of lipid degradation. The findings have important implications to the understanding of the stability of the myelin membrane.     

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ～1 μM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1β. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.     

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.     

HIV gp41 Fusion Peptide Increases Membrane Ordering in a Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.     

Vaccinia virus replication is not affected by APOBEC3 family members

Background

HIV-1 entry into cells is a multifaceted process involving target cell CD4 and the chemokine receptors, CXCR4 or CCR5. The lipid composition of the host cell plays a significant role in the HIV fusion process as it orchestrates the appropriate disposition of CD4 and co-receptors required for HIV-1 envelope glycoprotein (Env)-mediated fusion. The cell membrane is primarily composed of sphingolipids and cholesterol. The effects of lipid modulation on CD4 disposition in the membrane and their role in HIV-1 entry have extensively been studied. To focus on the role of lipid composition on chemokine receptor function, we have by-passed the CD4 requirement for HIV-1 Env-mediated fusion by using a CD4-independent strain of HIV-1 Env.

Results

Cell fusion mediated by a CD4-independent strain of HIV-1 Env was monitored by observing dye transfer between Env-expressing cells and NIH3T3 cells bearing CXCR4 or CCR5 in the presence or absence of CD4. Chemokine receptor signaling was assessed by monitoring changes in intracellular [Ca²⁺] mobilization induced by CCR5 or CXCR4 ligand. To modulate target membrane cholesterol or sphingolipids we used Methyl-β-cyclodextrin (MβCD) or 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), respectively. Treatment of the target cells with these agents did not change the levels of CD4 or CXCR4, but reduced levels of CCR5 on the cell surface. Chemokine receptor signalling was inhibited by cholesterol removal but not by treatment with PPMP. HIV-1 Env mediated fusion was inhibited by >50% by cholesterol removal. Overall, PPMP treatment appeared to slow down the rates of CD4-independent HIV-1 Env-mediated Fusion. However, in the case of CXCR4-dependent fusion, the differences between untreated and PPMP-treated cells did not appear to be significant.

Conclusion

Although modulation of cholesterol and sphingolipids has similar effects on CD4 -dependent HIV-1 Env-mediated fusion, sphingolipid modulation had little effect on CD4-independent HIV-1 Env-mediated fusion. Chemokine receptor function remained intact following treatment of cells with PPMP. Therefore such treatment may be considered a more suitable agent to inhibit CD4 dependent HIV-1 infection.     

High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays.     

Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.     

Determinants of membrane activity from mutational analysis of the HIV fusion peptide

We have synthesized a small library of 38 variants of the 23-residue fusion peptide domain found at the N-terminus of gp41 glycoprotein of HIV. This hydrophobic, glycine-rich sequence is critical for viral infectivity and is thought to be central in the membrane fusion of viral envelope with the host membrane. There has been extensive discussion regarding the origin of fusogenicity in this viral fusion sequence. Our library of fusion peptide variants was designed to address the biophysical importance of secondary structure, peptide flexibility, glycine content, and placement. We assayed each peptide for its ability to induce lipid mixing and membrane permeablization in synthetic vesicles. We find that the viral fusion peptide may be greatly simplified while retaining fusogenic function and minimizing membrane-permeablizing function; to the best of our knowledge, this is the first attempt to optimize fusogenic function of the HIV fusion peptide through sequence variation. Our data show that many flexible, linear, minimally hydrophobic peptides may achieve the biophysical function of fusion; glycine does not appear to be essential. These findings will be useful in the design of synthetic fusogens for cellular delivery.     

Secretory vesicle cholesterol: Correlating lipid domain organization and Ca2 + triggered fusion

Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca^2 + sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure–area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal–spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca^2 + triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure.     

HIV gp41 Fusion Peptide Increases Membrane Ordering in a?Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.     

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.     

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.     

How calcium may cause exocytosis in sea urchin eggs

The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosisin vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipid can induce a phase transition that will promote fusion of apposed lipid bilayers. The process of exocytosis involves the secretory granule core as well as the lipids of the membrane. The osmotic properties of the granule contents lead to swelling of the granule during exocytosis. Swelling promotes the dispersal of the contents as they are extruded through the exocytotic pore. The movements of water and ions during exocytosis may also stabilize the transient fusion intermediate and consolidate the exocytotic pore as fusion occurs.