Intra-protein interacting collective modes in the terahertz frequency region.

Understanding how proteins work requires a thorough understanding of their internal dynamics. Proteins support a wide range of motions, from the femtoseconds to seconds time scale, relevant to crucial biological functions. In this context, the term "protein collective dynamics" refers to the complex patterns of coordinated motions of numerous atoms throughout the protein in the sub-picosecond time scale (terahertz frequency region). It is hypothesized that these dynamics have a substantial impact on the regulation of functional dynamical mechanisms, including ligand binding and allosteric signalling, charge transport direction, and the regulation of thermodynamic and thermal transport properties. Using the theoretical framework of hydrodynamics, the collective dynamics of proteins had previously been described in a manner akin to that of simple liquids, i.e. in terms of a single acoustic-like excitation, related to intra-protein vibrational motions. Here, we employ an interacting-mode model to analyse the results from molecular dynamics simulations and we unveil that the vibrational landscape of proteins is populated by multiple acoustic-like and low-frequency optic-like modes, with mixed symmetry and interfering with each other. We propose an interpretation at the molecular level of the observed scenario that we relate to the side-chains and the hydrogen-bonded networks dynamics. The present insights provide a perspective for understanding the molecular mechanisms underlying the energy redistribution processes in the interior of proteins.

Atorvastatin Modulates the Efficacy of Electroporation and Calcium Electrochemotherapy.

Electroporation is influenced by the features of the targeted cell membranes, e.g., the cholesterol content and the surface tension of the membrane. The latter is eventually affected by the organization of actin fibers. Atorvastatin is a statin known to influence both the cholesterol content and the organization of actin. This work analyzes the effects of the latter on the efficacy of electroporation of cancer cells. In addition, herein, electroporation was combined with calcium chloride (CaEP) to assess as well the effects of the statin on the efficacy of electrochemotherapy. Cholesterol-rich cell lines MDA-MB231, DU 145, and A375 underwent (1) 48 h preincubation or (2) direct treatment with 50 nM atorvastatin. We studied the impact of the statin on cholesterol and actin fiber organization and analyzed the cells' membrane permeability. The viability of cells subjected to PEF (pulsed electric field) treatments and CaEP with 5 mM CaCl2 was examined. Finally, to assess the safety of the therapy, we analyzed the N-and E-cadherin localization using confocal laser microscopy. The results of our investigation revealed that depending on the cell line, atorvastatin preincubation decreases the total cholesterol in the steroidogenic cells and induces reorganization of actin nearby the cell membrane. Under low voltage PEFs, actin reorganization is responsible for the increase in the electroporation threshold. However, when subject to high voltage PEF, the lipid composition of the cell membrane becomes the regulatory factor. Namely, preincubation with atorvastatin reduces the cytotoxic effect of low voltage pulses and enhances the cytotoxicity and cellular changes induced by high voltage pulses. The study confirms that the surface tension regulates of membrane permeability under low voltage PEF treatment. Accordingly, to reduce the unfavorable effects of preincubation with atorvastatin, electroporation of steroidogenic cells should be performed at high voltage and combined with a calcium supply.

Lipid composition of the cancer cell membrane.

Cancer cell possesses numerous adaptations to resist the immune system response and chemotherapy. One of the most significant properties of the neoplastic cells is the altered lipid metabolism, and consequently, the abnormal cell membrane composition. Like in the case of phosphatidylcholine, these changes result in the modulation of certain enzymes and accumulation of energetic material, which could be used for a higher proliferation rate. The changes are so prominent, that some lipids, such as phosphatidylserines, could even be considered as the cancer biomarkers. Additionally, some changes of biophysical properties of cell membranes lead to the higher resistance to chemotherapy, and finally to the disturbances in signalling pathways. Namely, the increased levels of certain lipids, like for instance phosphatidylserine, lead to the attenuation of the immune system response. Also, changes in lipid saturation prevent the cells from demanding conditions of the microenvironment. Particularly interesting is the significance of cell membrane cholesterol content in the modulation of metastasis. This review paper discusses the roles of each lipid type in cancer physiology. The review combined theoretical data with clinical studies to show novel therapeutic options concerning the modulation of cell membranes in oncology.

In Vitro Study of Calcium Microsecond Electroporation of Prostate Adenocarcinoma Cells.

Electroporation, applied as a non-thermal ablation method has proven to be effective for focal prostate treatment. In this study, we performed pre-clinical research, which aims at exploring the specific impact of this so-called calcium electroporation on prostate cancer. First, in an in-vitro study of DU 145 cell lines, microsecond electroporation (µsEP) parameters were optimized. We determined hence the voltage that provides both high permeability and viability of these prostate cancer cells. Subsequently, we compared the effect of µsEP on cells' viability with and without calcium administration. For high-voltage pulses, the cell death's mechanism was evaluated using flow-cytometry and confocal laser microscopy. For lower-voltage pulses, the influence of electroporation on prostate cancer cell mobility was studied using scratch assays. Additionally, we applied calcium-binding fluorescence dye (Fluo-8) to observe the calcium uptake dynamic with the fluorescence microscopy. Moreover, the molecular dynamics simulation visualized the process of calcium ions inflow during µsEP. According to our results calcium electroporation significantly decreases the cells viability by promoting apoptosis. Furthermore, our data shows that the application of pulsed electric fields disassembles the actin cytoskeleton and influences the prostate cancer cells' mobility.

Oxidative Effects during Irreversible Electroporation of Melanoma Cells-In Vitro Study.

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2-4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2-0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2-0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids' oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.

Cepharanthine induces ROS stress in glioma and neuronal cells via modulation of VDAC permeability.

Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid. Molecular dynamics studies show that CEP interacts with Voltage-dependent anion channel (VDAC), inducing the voltage-independent channel narrowing. In the new conformation, transport between mitochondria and cytoplasm is altered, which leads to the dose-dependent cytotoxicity. The biological effects of the interaction were investigated on glioblastoma multiforme (SNB-19) and neuronal (PC-12 + NGF) cell lines. The cytotoxic potential of cepharanthine was determined by MTT assay and flow cytometry apoptosis/necrosis studies. T-type calcium channel and VDAC were labelled by the immunocytochemical method. Additionally, fluorescent labelling of reactive oxygen species and mitochondria was performed. Changes in the pore size of VDAC were calculated as well. Molecular dynamics simulations were carried out to examine the interactions of cepharanthine with VDAC. The obtained results prove that cepharanthine enhances the apoptosis in glioma and neuronal cells by the release of reactive oxygen species. Cepharanthine alters the mitochondria-to-cytoplasm transport and thus induces the cytotoxicity with no selectivity.

Effects of hydration on the protonation state of a lysine analog crossing a phospholipid bilayer - insights from molecular dynamics and free-energy calculations.

The low bioavailability of most therapeutic compounds is often counterbalanced by association with molecular vectors capable of crossing cell membranes. Previous studies demonstrated that for vectors bearing titratable chemical groups, the translocation process might be accompanied by a change in the protonation state. For simple compounds e.g. a lysine analog, free energy calculations, using a single collective variable, namely the insertion depth, suggest that such a transition could only take place if the amino acid diffuses deep enough into the hydrophobic core of the membrane, a situation thermodynamically unfavorable. Here, we determined the 2D potential of mean force associated with the translocation of lysine across a model membrane using as reaction coordinates not only its location in the bilayer but also its hydration. Our results cogently demonstrate that the change in protonation can result from a small fluctuation in the latter, even at low insertion depth.

Free-energy landscape of ion-channel voltage-sensor-domain activation.

Voltage sensor domains (VSDs) are membrane-bound protein modules that confer voltage sensitivity to membrane proteins. VSDs sense changes in the transmembrane voltage and convert the electrical signal into a conformational change called activation. Activation involves a reorganization of the membrane protein charges that is detected experimentally as transient currents. These so-called gating currents have been investigated extensively within the theoretical framework of so-called discrete-state Markov models (DMMs), whereby activation is conceptualized as a series of transitions across a discrete set of states. Historically, the interpretation of DMM transition rates in terms of transition state theory has been instrumental in shaping our view of the activation process, whose free-energy profile is currently envisioned as composed of a few local minima separated by steep barriers. Here we use atomistic level modeling and well-tempered metadynamics to calculate the configurational free energy along a single transition from first principles. We show that this transition is intrinsically multidimensional and described by a rough free-energy landscape. Remarkably, a coarse-grained description of the system, based on the use of the gating charge as reaction coordinate, reveals a smooth profile with a single barrier, consistent with phenomenological models. Our results bridge the gap between microscopic and macroscopic descriptions of activation dynamics and show that choosing the gating charge as reaction coordinate masks the topological complexity of the network of microstates participating in the transition. Importantly, full characterization of the latter is a prerequisite to rationalize modulation of this process by lipids, toxins, drugs, and genetic mutations.

A molecular insight into the electro-transfer of small molecules through electropores driven by electric fields.

The transport of chemical compounds across the plasma membrane into the cell is relevant for several biological and medical applications. One of the most efficient techniques to enhance this uptake is reversible electroporation. Nevertheless, the detailed molecular mechanism of transport of chemical species (dyes, drugs, genetic materials, …) following the application of electric pulses is not yet fully elucidated. In the past decade, molecular dynamics (MD) simulations have been conducted to model the effect of pulsed electric fields on membranes, describing several aspects of this phenomenon. Here, we first present a comprehensive review of the results obtained so far modeling the electroporation of lipid membranes, then we extend these findings to study the electrotransfer across lipid bilayers subject to microsecond pulsed electric fields of Tat11, a small hydrophilic charged peptide, and of siRNA. We use in particular a MD simulation protocol that allows to characterize the transport of charged species through stable pores. Unexpectedly, our results show that for an electroporated bilayer subject to transmembrane voltages in the order of 500mV, i.e. consistent with experimental conditions, both Tat11 and siRNA can translocate through nanoelectropores within tens of ns. We discuss these results in comparison to experiments in order to rationalize the mechanism of drug uptake by cells. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Atomistic Simulations of Electroporation of Model Cell Membranes.

Electroporation is a phenomenon that modifies the fundamental function of the cell since it perturbs transiently or permanently the integrity of its membrane. Today, this technique is applied in fields ranging from biology and biotechnology to medicine, e.g., for drug and gene delivery into cells, tumor therapy, etc., in which it made it to preclinical and clinical treatments. Experimentally, due to the complexity and heterogeneity of cell membranes, it is difficult to provide a description of the electroporation phenomenon in terms of atomically resolved structural and dynamical processes, a prerequisite to optimize its use. Atomistic modeling in general and molecular dynamics (MD) simulations in particular have proven to be an effective approach for providing such a level of detail. This chapter provides the reader with a comprehensive account of recent advances in using such a technique to complement conventional experimental approaches in characterizing several aspects of cell membranes electroporation.

Voltage-gated ion channel modulation by lipids: insights from molecular dynamics simulations.

Cells commonly use lipids to modulate the function of ion channels. The lipid content influences the amplitude of the ionic current and changes the probability of voltage-gated ion channels being in the active or in the resting states. Experimental findings inferred from a variety of techniques and molecular dynamics studies have revealed a direct interaction between the lipid headgroups and the ion channel residues, suggesting an influence on the ion channel function. On the other hand the alteration of the lipids may in principle modify the overall electrostatic environment of the channel, and hence the transmembrane potential, leading to an indirect modulation, i.e. a global effect. Here we have investigated the structural and dynamical properties of the voltage-gated potassium channel Kv1.2 embedded in bilayers with modified upper or lower leaflet compositions corresponding to realistic biological scenarios: the first relates to the effects of sphingomyelinase, an enzyme that modifies the composition of lipids of the outer membrane leaflets, and the second to the effect of the presence of a small fraction of PIP2, a highly negatively charged lipid known to modulate voltage-gated channel function. Our molecular dynamics simulations do not enable to exclude the global effect mechanism in the former case. For the latter, however, it is shown that local interactions between the ion channel and the lipid headgroups are key-elements of the modulation.

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory.

Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Omega currents in voltage-gated ion channels: what can we learn from uncovering the voltage-sensing mechanism using MD simulations?

Ion channels conduct charged species through otherwise impermeable biological membranes. Their activity supports a number of physiological processes, and genetic mutations can disrupt their function dramatically. Among these channels, voltage gated cation channels (VGCCs) are ubiquitous transmembrane proteins involved in electrical signaling. In addition to their selectivity for ions, their function requires membrane-polarization-dependent gating. Triggered by changes in the transmembrane voltage, the activation and deactivation of VGCCs proceed through a sensing mechanism that prompts motion of conserved positively charged (basic) residues within the S4 helix of a four-helix bundle, the voltage sensor domain (VSD). Decades of experimental investigations, using electrophysiology, molecular biology, pharmacology, and spectroscopy, have revealed details about the function of VGCCs. However, in 2005, the resolution of the crystal structure of the activated state of one member of the mammalian voltage gated potassium (Kv) channels family (the Kv1.2) enabled researchers to make significant progress in understanding the structure-function relationship in these proteins on a molecular level. In this Account, we review the use of a complementary technique, molecular dynamics (MD) simulations, that has offered new insights on this timely issue. Starting from the "open-activated state" crystal structure, we have carried out large-scale all atom MD simulations of the Kv1.2 channel embedded in its lipidic environment and submitted to a hyperpolarizing (negative) transmembrane potential. We then used steered MD simulations to complete the full transition to the resting-closed state. Using these procedures, we have followed the operation of the VSDs and uncovered three intermediate states between their activated and deactivated conformations. Each conformational state is characterized by its network of salt bridges and by the occupation of the gating charge transfer center by a specific S4 basic residue. Overall, the global deactivation mechanism that we have uncovered agrees with proposed kinetic models and recent experimental results that point towards the presence of several intermediate states. The understanding of these conformations has allowed us to examine how mutations of the S4 basic residues analogous to those involved in genetic diseases affect the function of VGCCs. In agreement with electrophysiology experiments, mutations perturb the VSD structure and trigger the appearance of state-dependent "leak" currents. The simulation results unveil the key elementary molecular processes involved in these so-called "omega" currents. We generalize these observations to other members of the VGCC family, indicating which type of residues may generate such currents and which conditions might cause leaks that prevent proper function of the channel. Today, the understanding of the intermediate state conformations enables researchers to confidently tackle other key questions such as the mode of action of toxins or modulation of channel function by lipids.

High-yield nontoxic gene transfer through conjugation of the CM18-Tat11 chimeric peptide with nanosecond electric pulses.

We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols.

Structural properties of archaeal lipid bilayers: small-angle X-ray scattering and molecular dynamics simulation study.

Aeropyrum pernix is an aerobic hyperthermophilic archaeon that grows in harsh environmental conditions and as such possesses unique structural and metabolic features. Its membrane interfaces with the extreme environment and is the first line of defense from external factors. Therefore, lipids composing this membrane have special moieties that increase its stability. The membrane of A. pernix is composed predominantly of two polar lipids 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI). Both have methyl branches in their lipid tails and ether linkages and carbohydrates in their headgroup. These moieties significantly affect the structure and dynamics of the bilayer. To provide a molecular level insight into these characteristics, we used here Molecular Dynamics (MD) simulations of lipid bilayers of composition similar to those of the archaeal membranes. First, we show that the electron density profiles along the normal to the bilayers derived from the simulations are in good agreement with the profiles obtained by the small-angle X-ray scattering (SAXS) technique, which provides confidence in the force fields used. Analyses of the simulation data show that the archaeal lipid bilayers are less hydrated than conventional phosphatidylcholine (PC) lipids and that their structure is not affected by the salt present in the surrounding solution. Furthermore, the lateral pressure in their hydrophobic core, due to the presence of the branched tails, is much higher than that at PC-based lipid bilayers. Both the methyl branched tails and the special headgroup moieties contribute to slow drastically the lateral diffusion of the lipids. Furthermore, we found that the lipid head groups associate via hydrogen bonding, which affects their reorientational dynamics. All together, our data provide links between the microscopic properties of these membranes and their overall stability in harsh environments.

Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations.

The response of a membrane-bound Kv1.2 ion channel to an applied transmembrane potential has been studied using molecular dynamics simulations. Channel deactivation is shown to involve three intermediate states of the voltage sensor domain (VSD), and concomitant movement of helix S4 charges 10-15 Å along the bilayer normal; the latter being enabled by zipper-like sequential pairing of S4 basic residues with neighboring VSD acidic residues and membrane-lipid head groups. During the observed sequential transitions S4 basic residues pass through the recently discovered charge transfer center with its conserved phenylalanine residue, F(233). Analysis indicates that the local electric field within the VSD is focused near the F(233) residue and that it remains essentially unaltered during the entire process. Overall, the present computations provide an atomistic description of VSD response to hyperpolarization, add support to the sliding helix model, and capture essential features inferred from a variety of recent experiments.

Structural effects of charge destabilization and amino acid substitutions in amyloid fragments of CsgA.

The most studied functional amyloid is the CsgA, major curli subunit protein, which is produced by numerous strains of Enterobacteriaceae. Although CsgA sequences are highly conserved, they exhibit species diversity, which reflects the specific evolutionary and functional adaptability of the major curli subunit. Herein, we performed bioinformatics analyses to uncover the differences in the amyloidogenic properties of the R4 fragments in Escherichia coli and Salmonella enterica and proposed four mutants for more detailed studies: M1, M2, M3, and M4. The mutated sequences were characterized by various experimental techniques, such as circular dichroism, ATR-FTIR, FT-Raman, thioflavin T, transmission electron microscopy and confocal microscopy. Additionally, molecular dynamics simulations were performed to determine the role of buffer ions in the aggregation process. Our results demonstrated that the aggregation kinetics, fibril morphology, and overall structure of the peptide were significantly affected by the positions of charged amino acids within the repeat sequences of CsgA. Notably, substituting glycine with lysine resulted in the formation of distinctive spherically packed globular aggregates. The differences in morphology observed are attributed to the influence of phosphate ions, which disrupt the local electrostatic interaction network of the polypeptide chains. This study provides knowledge on the preferential formation of amyloid fibrils based on charge states within the polypeptide chain.

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Dual effect of phosphatidylinositol (4,5)-bisphosphate PIP(2) on Shaker K(+) [corrected] channels.

Phosphatidylinositol (4,5)-bisphosphate (PIP(2)) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP(2) on the open state stabilization. A similar effect of PIP(2) on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K(+) channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP(2) effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP(2) exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

On the electroporation thresholds of lipid bilayers: molecular dynamics simulation investigations.

Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the "electrical" properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.