Spontaneous Pore Research Articles

Optimal conditions for opening of membrane pore by amphiphilic peptides.

Amphiphilic peptides can interact with biological membranes and severely affect their barrier and signaling functions. These peptides, including antimicrobial peptides, can self-assemble into transmembrane pores that cause cell death. Despite their medical importance, the conditions required for pore formation remain elusive. Monte Carlo simulations with coarse-grained models enabled us to calculate the free energies of pore opening under various conditions. In agreement with oriented circular dichroism experiments, a high peptide-to-lipid ratio was found to be necessary for spontaneous pore assembly. The peptide length has a non-monotonic impact on pore formation, and the optimal length matches with the membrane thickness. Furthermore, the hydrophobicity of the peptide ends and the mutual positions of peptides on the membrane play a role.

Influence of H2TOEtPyP4 porphyrin on the stability and conductivity of bilayer lipid membranes.

Many water-soluble cationic porphyrins are known to be prospective chemotherapeutics and photosensitizers for cancer treatment and diagnosis. The physicochemical properties of porphyrins, in particular their interactions with membranes, are important determining factors of their biological activity. The influence of cationic meso-tetra-[4-N-(2'-hydroxyethyl) pyridyl] porphyrin (H2TOEtPyP) on the stability and conductivity of bilayer lipid membranes (BLMs) was studied. H2TOEtPyP4 porphyrin was shown to decrease the stability of BLMs made of a mixture of DOPS and DPPE (1:1) in an electric field because of a reduction of line tension of spontaneously formed pore edges in the BLM. The presence of cationic porphyrin was found to reduce BLM surface tension. This effect was enhanced with increasing porphyrin concentration. H2TOEtPyP4 increased the probability of spontaneous pore formation. Further investigating the cyclic current-voltage characteristics of BLMs allowed determining the electrical capacity and conductivity of BLMs in the presence of H2TOEtPyP4 porphyrin. It was shown that in the presence of cationic porphyrin the electrical capacity as well as conductivity of the BLM increases.

Pore Formation Mechanisms of Melittin-Like Membrane-Active Peptides

Pore formation by membrane-active peptides is believed to play a key role in the mechanism of action of antimicrobial peptides. However, at present the process of spontaneous pore formation, the pore structure, and the pore lifetime in the bilayer remain unknown. This is largely due to experimental difficulties in resolving transient protein structures and dynamics in fluid-phase lipid bilayers. Here we employ a mixture of unbiased multi-microsecond molecular dynamics simulations and experimental techniques to study the process of pore formation and conductance of melittin, a pore-forming membrane active peptide from honey-bee venom, as well as synthetically evolved gain-of-function variants of melittin, which have recently been developed by William Wimley at Tulane University.Using spectroscopic techniques we show that gain-of-function variants are able to leak larger dyes from unilamellar vesicles than melittin. Like melittin gain-of-function variants result in virtually complete leakage of the dye from vesicles. However, the concentration of peptide required to achieve leakage is significantly lower for gain-of-function variants than for melittin. This suggests that these peptides form larger pores than melittin and that these pores are much more stable, remaining functional over the lifetime of the leakage experiment. Simulations of both melittin and gain-of-function variants reveal a wealth of atomic detail information about transient processes such as peptide absorption, folding, and oligomeric assembly, as well as the equilibrium structural ensemble and stability, which were verified using circular dichroism, fluorescence, and electrochemical impedance spectroscopy.

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo.

Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover. Here we use morphological and molecular in vivo imaging after contusion SCI in mice to show that injured axons persist in a metastable state for hours. Intra-axonal calcium dynamics influence fate, but the outcome is not invariably destructive in that many axons with calcium elevations recover homeostasis without intervention. Calcium enters axons primarily through mechanopores. Spontaneous pore resealing allows calcium levels to normalize and axons to survive long term. Axon loss can be halted by blocking calcium influx or calpain, even with delayed initiation. Our data identify an inherent self-preservation process in contused axons and a window of opportunity for rescuing connectivity after nontransecting SCI.

Rupture of lipid vesicles near solid surfaces.

The behavior of lipid vesicles near solid surfaces, despite its scientific and technological significance, is poorly understood. By simultaneously taking into account (i) the dynamics of spontaneous pore opening and closing in surface bound vesicles; (ii) their volume loss via leakage through the pores; (iii) and the propagation of their contact line, we have developed a simple model that can fully describe the detailed mechanism of and provide the necessary conditions for the rupture of vesicles and the subsequent formation of supported lipid bilayers. The predictions of the model are in qualitative agreement with many of the experimental observations.

Simulations of the Rupture of Liposomes Near Solid Surfaces in the Presence of Membrane-Membrane Adhesion

The behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. In spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. Our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion (not only between the membrane and the surface, but also between two adjacent membrane areas), and finally spread along the surface forming a supported lipid bilayer. In our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion fronts. All these processes are supposed to follow an overdamped dynamics and are coupled to each other through membrane tension.Here we identify under which conditions the dynamics leads to the formation of hat shaped geometries with a projecting brim. We found that the most determining parameters with respect to the shape and dynamics of a vesicle are the drag coefficient along the adhesion front and the line tension along the pore. Using numerical simulations we could reproduce a variety of experimental observations and conclude that a long lasting hat shaped geometry occurs in a limited range of parameters.

Simulations of the Rupture of Liposomes Near Solid Surfaces

The behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. In spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. Our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion (not only between the membrane and the surface, but also between two adjacent membrane areas), and finally spread along the surface forming a supported lipid bilayer. In our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion fronts. All these processes are supposed to follow an overdamped dynamics and are coupled to each other through membrane tension. Here we identify under which conditions the dynamics leads to the formation of hat shaped geometries with a projecting brim, and compare our results with experimental observations.

Behavior of Lipid Vesicles Near Solid Surfaces

The behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. In spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. Our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. In our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. All these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension.Our numerical simulations uncover the nature of the entire rupture process, and make quantitative predictions about the conditions under which fast vesicle rupture and supported lipid bilayer formation is expected.

The effect of lipid oxidation on the water permeability of phospholipids bilayers

The effect of lipid oxidation on water permeability of phosphatidylcholine membranes was investigated by means of both scattering stopped flow experiments and atomistic molecular dynamics simulations. Formation of water pores followed by a significant enhancement of water permeability was observed. The molecules of oxidized phospholipids facilitate pore formation and subsequently stabilize water in the membrane interior. A wide range of oxidation ratios, from 15 to 100 mol%, was considered. The degree of oxidation was found to strongly influence the time needed for the opening of a pore. In simulations, the oxidation ratio of 75 mol% was found to be a threshold for spontaneous pore formation in the tens of nanosecond timescale, whereas 15 mol% of oxidation led to significant water permeation in the timescale of seconds. Once a pore was formed, the water permeability was found to be virtually independent of the oxidation ratio.

Toward understanding protocell mechanosensation.

Mechanosensitive (MS) channels can prevent bacterial bursting during hypo-osmotic shocks by responding to increases in lateral tension at the membrane level through an integrated and coordinated opening mechanism. Mechanical regulation in protocells could have been one of the first mechanisms to evolve in order to preserve their integrity against changing environmental conditions. How has the rich functional diversity found in present cells been created throughout evolution, and what did the primordial MS channels look like? This review has been written with the aim of identifying which factors may have been important for the appearance of the first osmotic valve in a prebiotic context, and what this valve may have been like. It highlights the mechanical properties of lipid bilayers, the association of peptides as aggregates in membranes, and the conservation of sequence motifs as central aspects to understand the evolution of proteins that gate below the tension required for spontaneous pore formation and membrane rupture. The arguments developed here apply to both MscL and MscS homologs, but could be valid to mechano-susceptible proteins in general.

Water Confinement in Hydrophobic Nanopores. Pressure-Induced Wetting and Drying

Wetting and drying of hydrophobic pores with diameters lower than 0.2 μm by aqueous solutions at different hydrostatic pressures is investigated by measuring the ionic conductance variation through the nanopores. The critical pressure for water intrusion into the nanopores increases with lowering the pore diameter and the surface tension of the hydrophobic modification, in agreement with the Laplace equation. Nevertheless, restoring the pressure to the atmospheric one does not result in spontaneous pore dewetting unless bubbles are left inside the pores. Such bubbles can appear at the regions of narrowing cross section and/or varying quality of the hydrophobic modification and thus can be engineered to control water expulsion.

Coupled-map-lattice model for spontaneous pore formation in anodic oxidation

We construct a coupled map lattice model for spontaneous pore formation in anodic oxidation in two dimensions and perform numerical simulations, after we explain steady flat solutions, their linear stability, and a single pore solution for a model of Parkhutik and Shershulsky.

The Temperature Dependence of Lipid Membrane Permeability, its Quantized Nature, and the Influence of Anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.

The Temperature Dependence And Quantized Nature Of The Lipid Membrane Permeability

We investigate the permeability of phospholipid membranes for fluorescence dyes and ions. We find that the permeability is strongly enhanced close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence Correlation Spectroscopy (FCS) reveals the permeability for rhodamine dyes across 100 nm vesicles. Using FCS, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events. Furthermore, we demonstrate that general anaesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anaesthetics decrease channel conductance and ultimately lead to ‘blocking’ of the lipid pores in experiments performed at or above the chain melting transition. Our data suggests that the macroscopic increase in permeability close to transitions and microscopic lipid channel formation are the same physical process.

155. Atomistic investigations of spontaneous unstable pore formation in DMPC lipid bilayers due to the presence of Me 2SO

155. Atomistic investigations of spontaneous unstable pore formation in DMPC lipid bilayers due to the presence of Me 2SO

Pore Formation Coupled to Ion Transport through Lipid Membranes as Induced by Transmembrane Ionic Charge Imbalance: Atomistic Molecular Dynamics Study

We have employed atomic-scale molecular dynamics simulations to address ion transport through transient water pores in phospholipid membranes. The formation of a water pore is induced by a transmembrane ionic charge imbalance, which gives rise to a significant potential difference across the membrane. The subsequent transport of ions through the pore discharges the transmembrane potential and makes the water pore metastable, leading eventually to its sealing. The findings highlight the importance of ionic charge fluctuations in spontaneous pore formation and their role in ion leakage through protein-free lipid membranes.

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells.

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.

Partition coefficients of alkanols in lipid bilayer/water

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence correlation spectroscopy reveals the permeability for rhodamine dyes across 100-nm vesicles. Using fluorescence correlation spectroscopy, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events very similar to those reported for channel proteins. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to blocking of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid ion channel formation are the same physical process.