Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the "free" patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.     

The exocytotic fusion pore interface: a model of the site of neurotransmitter release

infrastructurel techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysio-logical techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer. Fusion pore formation is regulated by a macromolecular scaffold of proteins that is responsible for bringing the plasma membrane into a highly curved dimple very close to a tense secretory granule membrane, creating the architecture where the strongly attractive hydrophobic force causes the membranes to form a ‘hemifusion’ intermediate. Membrane fusion is completed by the formation of an aqueous pore after rupture of the shared bilayer. We also propose that the microenvironment of the interface when the pore first opens, dominated by the charged groups on the secretory vesicle matrix and phospholipids, will greatly influence the release of secretory products.     

SNARE Complex Zipping as a Driving Force in the Dilation of Proteinaceous Fusion Pores

The assembly of SNARE proteins into a tight complex has been hypothesized to drive membrane fusion. A model of the initial fusion pore as a proteinaceous channel formed by SNARE proteins places their membrane anchors in separate membranes. This leaves the possibility of a final assembly step that brings the membrane anchors together and drives fusion pore expansion. The present study develops a model for expansion in which the final SNARE complex zipping step drives a transition from a proteinaceous fusion pore to a lipidic fusion pore. An estimate of the energy released upon merger of the helical segments of the SNARE motifs with the helical segments of the membrane anchors indicates that completing the assembly of a few SNARE complexes can overcome the elastic energy that opposes lipid bilayer deformation into a narrow fusion pore. The angle between the helical axes of the membrane anchor and SNARE motif serves as a useful reaction coordinate for this transition. Energy was calculated as a function of this angle, incorporating contributions from membrane bending, SNARE complex assembly, membrane anchor flexing and hydrophobic interactions. The rate of this transition was evaluated as a process of diffusion over the barrier imposed by these combined energies, and the rates estimated were consistent with experimental measurements.     

Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin

The antibacterial activity of many small, positively charged peptides and proteins is based on pore formation in lipid bilayers. It is here proposed to arise from an electroporation effect. This hypothesis is supported by calculations of the electrostatic potential of NK-lysin associated to a membrane. For a significant area of the protein-membrane interface, the electrostatic potential is found to be above the minimum threshold for electroporation. A single highly charged alpha-helical segment of NK-lysin is mainly responsible for this effect. It is experimentally demonstrated that a peptide comprising this helix has antibacterial activity. We propose that superficial association to membranes suffices to trigger electroporation, provided the peptide is sufficiently charged. The effect is referred to as molecular electroporation.     

Interaction of the antimicrobial peptide cyclo(RRWWRF) with membranes by molecular dynamics simulations

Antimicrobial peptides have gained a lot of interest in recent years due to their potential use as a new generation of antibiotics. It is believed that this type of relatively short, amphipathic, cationic peptide targets the bacterial membrane, and destroys the chemical gradients over the membrane via formation of stable or transient pores. Here we use the NMR structure of cyclo(RRWWRF) in a series of molecular dynamics simulations in membranes at various peptide/lipid ratios. We observe that the NMR structure of the peptide is still stable after 100 ns simulation. At a peptide/lipid ratio of 2:128, the membrane is only a little affected compared to a pure dipalmitoylphosphatidylcholine lipid membrane, but at a ratio of 12:128, the water-lipid interface becomes more fuzzy, the water molecules can reach deeper into the hydrophobic core, and the water penetration free-energy barrier changes. Moreover, we observe that the area per lipid decreases and the deuterium order parameters increase in the presence of the peptide. We suggest that the changes in the hydrophobic core, together with the changes in the headgroups, result in an imbalance of the membrane and that it is thus not an efficient hydrophobic barrier in the presence of the peptides, independent of pore formation.     

Pore formation by a Bax-derived peptide: effect on the line tension of the membrane probed by AFM

Bax is a critical regulator of physiological cell death that increases the permeability of the outer mitochondrial membrane and facilitates the release of the so-called apoptotic factors during apoptosis. The molecular mechanism of action is unknown, but it probably involves the formation of partially lipidic pores induced by Bax. To investigate the interaction of Bax with lipid membranes and the physical changes underlying the formation of Bax pores, we used an active peptide derived from helix 5 of this protein (Bax-alpha5) that is able to induce Bax-like pores in lipid bilayers. We report the decrease of line tension due to peptide binding both at the domain interface in phase-separated lipid bilayers and at the pore edge in atomic force microscopy film-rupture experiments. Such a decrease in line tension may be a general strategy of pore-forming peptides and proteins, as it affects the energetics of the pore and stabilizes the open state.     

Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion

We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.     

Lipid saturation and head group composition have a pronounced influence on the membrane insertion equilibrium of amphipathic helical polypeptides

The histidine-rich peptides of the LAH4 family were designed using cationic antimicrobial peptides such as magainin and PGLa as templates. The LAH4 amphipathic helical sequences exhibit a multitude of interesting biological properties such as antimicrobial activity, cell penetration of a large variety of cargo and lentiviral transduction enhancement. The parent peptide associates with lipid bilayers where it changes from an orientation along the membrane interface into a transmembrane configuration in a pH-dependent manner. Here we show that LAH4 adopts a transmembrane configuration in fully saturated DMPC membranes already at pH 3.5, i.e. much below the pK_a of the histidines whereas the transition pH in POPC correlates closely with histidine neutralization. In contrast in POPG membranes the in-planar configuration is stabilized by about one pH unit. The differences in pH can be converted into energetic contributions for the in-plane to transmembrane transition equilibrium, where the shift in the transition pH due to lipid saturation corresponds to energies which are otherwise obtained by the exchange of several cationic with hydrophobic residues. A similar dependence on lipid saturation has also been observed when the PGLa and magainin antimicrobial peptides interact within lipid bilayers suggesting that the quantitative evaluation presented in this paper also applies to other membrane polypeptides.     

Dynamics of fusion pores connecting membranes of different tensions

The energetics underlying the expansion of fusion pores connecting biological or lipid bilayer membranes is elucidated. The energetics necessary to deform membranes as the pore enlarges, in some combination with the action of the fusion proteins, must determine pore growth. The dynamics of pore growth is considered for the case of two homogeneous fusing membranes under different tensions. It is rigorously shown that pore growth can be quantitatively described by treating the pore as a quasiparticle that moves in a medium with a viscosity determined by that of the membranes. Motion is subject to tension, bending, and viscous forces. Pore dynamics and lipid flow through the pore were calculated using Lagrange's equations, with dissipation caused by intra- and intermonolayer friction. These calculations show that the energy barrier that restrains pore enlargement depends only on the sum of the tensions; a difference in tension between the fusing membranes is irrelevant. In contrast, lipid flux through the fusion pore depends on the tension difference but is independent of the sum. Thus pore growth is not affected by tension-driven lipid flux from one membrane to the other. The calculations of the present study explain how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. In a similar fashion, swelling of secretory granules after fusion in biological systems could promote pore enlargement during exocytosis. The calculations also show that pore expansion can be caused by pore lengthening; lengthening may be facilitated by fusion proteins.     

Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins

Pore-forming toxins (PFTs) are proteins that are secreted as soluble molecules and are inserted into membranes to form oligomeric transmembrane pores. In this paper, we report the crystal structure of Fragaceatoxin C (FraC), a PFT isolated from the sea anemone Actinia fragacea, at 1.8?? resolution. It consists of a crown-shaped nonamer with an external diameter of about 11.0?nm and an internal diameter of approximately 5.0?nm. Cryoelectron microscopy studies of FraC in lipid bilayers reveal the pore structure that traverses the membrane. The shape and dimensions of the crystallographic oligomer are fully consistent with the membrane pore. The FraC structure provides insight into the interactions governing the assembly process and suggests the structural changes that allow for membrane insertion. We propose a nonameric pore model that spans the membrane by forming a lipid-free α-helical bundle pore.     

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.     

Calculating Transition Energy Barriers and Characterizing Activation States for Steps of Fusion

We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.     

Regulation of transbilayer plasma membrane phospholipid asymmetry

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.     

Osmotically induced membrane tension modulates membrane permeabilization by class L amphipathic helical peptides: nucleation model of defect formation.

The mechanism of action of lytic peptides on membranes is widely studied and is important in view of potential medical applications. Previously (I. V. Polozov, A. I. Polozova, E. M. Tytler, G. M. Anantharamaiah, J. P. Segrest, G. A. Woolley, and R. M., Biochemistry, 36:9237--9245) we analyzed the mechanism of membrane permeabilization by 18L, the archetype lytic peptide featuring the class L amphipathic alpha-helix, according to the classification of Segrest et al. (J. P. Segrest, G. de Loof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah, 1990, Proteins, 8:103--117). We concluded that the 18L peptide destabilizes membranes, leading to a transient formation of large defects that result in contents leakage and, in the presence of bilayer-bilayer contact, could lead to vesicle fusion. Here we report that this defect formation is strongly enhanced by the membrane tension induced by osmotic swelling of vesicles. Even below standard leakage-inducing peptide/lipid ratios, membrane resistance to osmotic tension drops from hundreds to tens of milliosmoles. The actual decrease is dependent on the peptide/lipid ratio and on the type of lipid. We propose that under membrane tension a peptidic pore serves as a nucleation site for the transient formation of a lipidic pore. The tension is released upon pore expansion with inclusion of more peptides and lipids into the pore lining. This tension modulation of leakage was observed for other class L peptides (mastoparan, K18L) and thus may be of general applicability for the action of membrane active lytic peptides.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.     

Cooperative antimicrobial action of melittin on lipid membranes: A coarse-grained molecular dynamics study

We conducted a series of coarse-grained molecular dynamics (CG-MD) simulations to investigate the complicated actions of melittin, which is an antimicrobial peptide (AMP) derived from honey bee venom, on a lipid membrane. To accurately simulate the AMP action, we developed and used a protein CG model as an extension of the pSPICA force field (FF), which was designed to reproduce several thermodynamic quantities and structural properties. At a low peptide-to-lipid (P/L) ratio (1/102), no defect was detected. At P/L = 1/51, toroidal pore formation was observed due to collective insertion of multiple melittin peptides from the N-termini. The pore formation was initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. At a higher P/L ratio (1/26), two more modes were detected, seemingly not controlled by the P/L ratio but by a local arrangement of melittin peptides: 1. Pore formation accompanied by lipid extraction by melittin peptides:a detergent-like mechanism. 2. A rapidly formed large pore in a significantly curved membrane: bursting. Thus, we observed three pore formation modes (toroidal pore formation, lipid extraction, and bursting) depending on the peptide concentration and local arrangement. These observations were consistent with experimental observations and hypothesized melittin modes. Through this study, we found that the local arrangements and population of melittin peptides and the area expansion rate by membrane deformation were key to the initiation of and competition among the multiple pore formation mechanisms.     

Incorporation of a polypeptide segment into the beta-domain pore during the assembly of a bacterial autotransporter.

Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'beta-domain' that forms a beta-barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the beta-domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the beta-domain pore. By examining the accessibility of tobacco etch virus protease sites and single-cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre-translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP beta-domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre-formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.     

Incorporation of a polypeptide segment into the β-domain pore during the assembly of a bacterial autotransporter

Bacterial autotransporters consist of an N-terminal 'passenger domain' that is transported into the extracellular space by an unknown mechanism and a C-terminal 'β-domain' that forms a β-barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the β-domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the β-domain pore. By examining the accessibility of tobacco etch virus protease sites and single-cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre-translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP β-domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre-formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.     

Gating of MscL studied by steered molecular dynamics

Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel. A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface. The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation. A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening. The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices. The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL. Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening.