Membrane permeability is one of the main determinants for the absorption, distribution, metabolism and excretion of compounds and is therefore of crucial importance for successful drug development. Experiments with artificial phospholipid membranes have shown that the intrinsic membrane permeability (P0) of compounds is well-predicted by the solubility-diffusion model (SDM). However, using the solubility-diffusion model to predict the P0 of biological Caco-2 and MDCK cell membranes has proven unreliable so far. Recent publications revealed that many published P0 extracted from Caco-2 and MDCK experiments are incorrect. In this work, we therefore used a small self-generated set as well as a large revised set of experimental Caco-2 and MDCK data from literature to compare experimental and predicted P0. The P0 extracted from Caco-2 and MDCK experiments were systematically lower than the P0 predicted by the solubility-diffusion model. However, using the following correlation: log P0,Caco-2/MDCK = 0.84 log P0,SDM - 1.85, P0 of biological Caco-2 and MDCK cell membranes was well-predicted by the solubility-diffusion model.

The cells of living organisms are surrounded by the biological membranes that form a barrier between the internal and external environment of the cells. Cell membranes serve as barriers and gatekeepers. They protect cells against the entry of undesirable substances and are the first line of interaction with foreign particles. Therefore, it is very important to understand how substances such as particulate matter (PM) interact with cell membranes. To investigate the effect of PM on the electrical properties of biological membranes, a series of experiments using a black lipid membrane (BLM) technique were performed. L-α-Phosphatidylcholine from soybean (azolectin) was used to create lipid bilayers. PM samples of different diameters (<4 (SRM-PM4.0) and <10 μm (SRM-PM10) were purchased from The National Institute of Standards and Technology (USA) to ensure the repeatability of the measurements. Lipid membranes with incorporated gramicidin A (5 pg/mL) ion channels were used to investigate the effect of PM on ion transport. The ionic current passing through the azolectin membranes was measured in ionic gradients (50/150 mM KCl on cis/trans side). In parallel, the electric membrane capacitance measurements, analysis of the conductance and reversal potential were performed. Our results have shown that PM at concentration range from 10 to 150 μg/mL reduced the basal ionic current at negative potentials while increased it at positive ones, indicating the interaction between lipids forming the membrane and PM. Additionally, PM decreased the gramicidin A channel activity. At the same time, the amplitude of channel openings as well as single channel conductance and reversal potential remained unchanged. Lastly, particulate matter at a concentration of 150 μg/mL did not affect the electric membrane capacity to any significant extent. Understanding the interaction between PM and biological membranes could aid in the search for effective cytoprotective strategies. Perhaps, by the use of an artificial system, we will learn to support the consequences of PM-induced damage.

Transwell experiments with Caco-2 or MDCK cells are the gold standard for determining the intestinal permeability of chemicals. The intrinsic membrane permeability (P0), that can be extracted from these experiments, might be comparable to P0 measured in black lipid membrane (BLM) experiments and P0 predicted by the solubility-diffusion model. Unfortunately, the overlap between experimental P0,Caco-2/MDCK and P0,BLM data is very small. So far, differences between both approaches have been attributed to the cholesterol and sphingomyelin content of cell membranes, but the database is too sparse to thoroughly test this theory. To create a diverse dataset, we measured P0,BLM of ten chemicals in BLM experiments using DPhPC and DPhPC/cholesterol/sphingomyelin membranes. The results were compared to predicted BLM data and experimental Caco-2/MDCK data obtained from literature. While P0,BLM of all chemicals was well predicted by the solubility-diffusion model, P0,Caco-2/MDCK was only predictable for rather hydrophilic compounds with logarithmic hexadecane/water partition coefficients below -0.5. The effect of cholesterol and sphingomyelin on P0,BLM was negligibly small.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE-PLFE and PLFE-substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Vibrio cholerae is a Gram-negative, facultative anaerobic bacterial species that causes serious disease and can grow on various carbon sources, including chitin polysaccharides. In saltwater, its attachment to chitin surfaces not only serves as the initial step of nutrient recruitment but is also a crucial mechanism underlying cholera epidemics. In this study, we report the first characterization of a chitooligosaccharide-specific chitoporin, VcChiP, from the cell envelope of the V. cholerae type strain O1. We modeled the structure of VcChiP, revealing a trimeric cylinder that forms single channels in phospholipid bilayers. The membrane-reconstituted VcChiP channel was highly dynamic and voltage induced. Substate openings O1\', O2\', and O3\', between the fully open states O1, O2, and O3, were polarity selective, with nonohmic conductance profiles. Results of liposome-swelling assays suggested that VcChiP can transport monosaccharides, as well as chitooligosaccharides, but not other oligosaccharides. Of note, an outer-membrane porin (omp)-deficient strain of Escherichia coli expressing heterologous VcChiP could grow on M9 minimal medium supplemented with small chitooligosaccharides. These results support a crucial role of chitoporin in the adaptive survival of bacteria on chitinous nutrients. Our findings also suggest a promising means of vaccine development based on surface-exposed outer-membrane proteins and the design of novel anticholera agents based on chitooligosaccharide-mimicking analogs.

Doxorubicin is an anti-cancer drug that is important for breast cancer therapy. In this study, the effects of the membrane potential of breast cancer cells (-30 mV) and normal breast epithelial cells (-60 mV) on doxorubicin (DOX) permeability was studied. To achieve this goal, black lipid membranes (BLMs) as a model cell membrane were formed with DPhPC phospholipids in a single aperture of a Teflon sheet by the Montal and Mueller method. The presence of the BLM was characterized by capacitive measurements. The measured specific capacitance of 0.6 µF/cm2 after applying the Montal and Mueller method, confirming the presence of a BLM in the aperture. In addition, the very low current leakage of the BLM (9-24 pA) and ClyA-protein channel insertion in the BLM indicate the compactness, high quality, and thickness of 3-5 nm of the BLM. Afterwards, the permeability of doxorubicin through the BLM was studied at defined cell conditions (37 °C and pH 7.4), as well as cancerous and healthy epithelial-cell membrane potentials (-30 mV and -60 mV, respectively). The results show a slow DOX penetration within the first few hours, which increases rapidly with time. The initial slow penetration can be attributed to an electrostatic interaction between doxorubicin and DPhPC molecules in the model cell membrane. Furthermore, a MTT assay on MCF-10A and MCF-7 under different concentrations of doxorubicin confirmed that the cancerous MCF-7 cell line is more resistant to doxorubicin in comparison with the non-cancerous MCF-10A. Such studies highlight important strategies for designing and tuning the interaction efficacy of novel pharmaceuticals at molecular level.

The main phospholipid (MPL) of Thermoplasma acidophilum DSM 1728 was isolated, purified and physico-chemically characterized by differential scanning calorimetry (DSC)/differential thermal analysis (DTA) for its thermotropic behavior, alone and in mixtures with other lipids, cholesterol, hydrophobic peptides and pore-forming ionophores. Model membranes from MPL were investigated; black lipid membrane, Langmuir-Blodgett monolayer, and liposomes. Laboratory results were compared to computer simulation. MPL forms stable and resistant liposomes with highly proton-impermeable membrane and mixes at certain degree with common bilayer-forming lipids. Monomeric bacteriorhodopsin and ATP synthase from Micrococcus luteus were co-reconstituted and light-driven ATP synthesis measured. This review reports about almost four decades of research on Thermoplasma membrane and its MPL as well as transfer of this research to Thermoplasma species recently isolated from Indonesian volcanoes.

Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia Here, we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1 M KCl at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 of K+/Cl- at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 to 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.IMPORTANCE Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. The large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, which are studied for the potential treatment of humans (2). Here, we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.

Interest in nanopore technology has been growing due to nanopores\' unique capabilities in small molecule sensing, measurement of protein folding, and low-cost DNA and RNA sequencing. The E. coli β-barrel outer membrane protein OmpG is an excellent alternative to other protein nanopores because of its single polypeptide chain. However, the flexibility of its extracellular loops ultimately limits applications in traditional biosensing. We deleted several residues in and near loop 6 of OmpG. The dynamic structure of the new construct determined by NMR shows that loops 1, 2, 6, and 7 have reduced flexibilities compared to those of wild-type. Electrophysiological measurements show that the new design virtually eliminates flickering between open and closed states across a wide pH range. Modification of the pore lumen with a copper chelating moiety facilitates detection of small molecules. As proof of concept, we demonstrate concurrent single-molecule biosensing of glutamate and adenosine triphosphate.

Black lipid membranes (BLMs) provide a biomimetic model system for studying cellular membrane processes, and are important tools in drug screening and biosensing applications. BLMs offer advantages over liposomes and solid-supported lipid bilayers in applications where access to both leaflets of the bilayer is critical. Reliable and repeatable formation of BLMs presents a major challenge, especially in systems that require interrogation of the membrane via optical microscopy. BLMs for optical interrogation are often formed by the manual painting method, which is tedious and has a high failure rate because it involves manual manipulation of nanoscale liquid films for membrane self-assembly. Here, we describe a fully automated technique for the formation of BLMs within the imaging plane of an inverted fluorescence microscope. The technique utilizes hydrostatic pressure manipulations within a simple microfluidic device, which are feedback controlled via confocal fluorescence monitoring of the BLM formation process. An algorithm for monitoring and precision control of BLM formation is devised and optimized to yield an 80% success rate for the formation of BLMs, with formation times on the order of 78 min. Membranes formed via the automated procedure are confirmed to be fluid and biomimetic via spontaneous insertion of α-hemolysin pores with characteristic conductance of ca. 1 nS. Graphical Abstract ᅟ.