Sterolysin from a 1950s culture of Karlodinium veneficum (aka Gymnodinium veneficum Ballantine) forms lethal sterol dependent membrane pores.

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957.

A Toxic Sterolysin From a 1950s Culture of Gymnodinium Veneficum Ballantine.

In 1957 Abbott and Ballentine described a highly toxic activity from a dinoflagellate isolated from the English Channel. in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (Abbotoxin and 59-E-Chloro-Abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to that described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and are consistent with the intial observations of Abbott and Ballentine from 1957.

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

The underlying mechanical properties of membranes tune their ability to fuse.

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aß) peptides in the brain. The processing of amyloid precursor protein (APP) into Aß is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aß1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Approaches for the modulation of mechanosensitive MscL channel pores.

MscL was the first mechanosensitive ion channel identified in bacteria. The channel opens its large pore when the turgor pressure of the cytoplasm increases close to the lytic limit of the cellular membrane. Despite their ubiquity across organisms, their importance in biological processes, and the likelihood that they are one of the oldest mechanisms of sensory activation in cells, the exact molecular mechanism by which these channels sense changes in lateral tension is not fully understood. Modulation of the channel has been key to understanding important aspects of the structure and function of MscL, but a lack of molecular triggers of these channels hindered early developments in the field. Initial attempts to activate mechanosensitive channels and stabilize functionally relevant expanded or open states relied on mutations and associated post-translational modifications that were often cysteine reactive. These sulfhydryl reagents positioned at key residues have allowed the engineering of MscL channels for biotechnological purposes. Other studies have modulated MscL by altering membrane properties, such as lipid composition and physical properties. More recently, a variety of structurally distinct agonists have been shown bind to MscL directly, close to a transmembrane pocket that has been shown to have an important role in channel mechanical gating. These agonists have the potential to be developed further into antimicrobial therapies that target MscL, by considering the structural landscape and properties of these pockets.

Selective Translocation of Cyclic Sugars through Dynamic Bacterial Transporter.

The selective translocation of molecules through membrane pores is an integral process in cells. We present a bacterial sugar transporter, CymA of unusual structural conformation due to a dynamic N terminus segment in the pore, reducing its diameter. We quantified the translocation kinetics of various cyclic sugars of different charge, size, and symmetry across native and truncated CymA devoid of the N terminus using single-channel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated pore with high affinity and translocate effectively. Specifically, these sugars bind and translocate rapidly through truncated CymA compared to native CymA. In contrast, larger cyclic heptasaccharides and octasaccharides do not translocate but bind to native and truncated CymA with distinct binding kinetics highlighting the importance of molecular charge, size and symmetry in translocation consistent with liposome assays. Based on the sugar-binding kinetics, we suggest that the N terminus most likely resides inside the native CymA barrel, regulating the transport rate of cyclic sugars. Finally, we present native CymA as a large nanopore sensor for the simultaneous single-molecule detection of various sugars at high resolution, establishing its functional versatility. This natural pore is expected to have several applications in nanobiotechnology and will help further our understanding of the fundamental mechanism of molecular transport.

A Role for Liquid-Ordered Plasma Membrane Nanodomains Coordinating the Unconventional Secretory Pathway of Fibroblast Growth Factor 2?

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that belongs to a subgroup of extracellular proteins lacking N-terminal signal peptides. Whereas this phenomenon was already recognized in the early 1990s, detailed insights into the molecular mechanisms underlying alternative pathways of protein secretion from eukaryotic cells were obtained only recently. Today, we know about a number of alternative secretory mechanisms, collectively termed unconventional protein secretion (UPS). FGF2 belongs to a subgroup of cargo proteins secreted by direct translocation across the plasma membrane. This feature has been classified as type I UPS and is shared with other unconventionally secreted proteins, such as HIV-Tat and Tau. FGF2 translocation across the membrane is initiated through sequential interactions with the Na,K-ATPase, Tec kinase, and phosphoinositide PI(4,5)P2 at the inner plasma membrane leaflet. Whereas the first two are auxiliary factors of this pathway, the interaction of FGF2 with PI(4,5)P2 triggers the core mechanism of FGF2 membrane translocation. It is based on a lipidic membrane pore that is formed by PI(4,5)P2-induced oligomerization of FGF2. Membrane-inserted FGF2 oligomers are recognized as translocation intermediates that are resolved at the outer plasma membrane leaflet by glypican-1, a heparan sulfate proteoglycan that captures and disassembles FGF2 oligomers on cell surfaces. Here, we discuss recent findings suggesting the molecular machinery mediating FGF2 membrane translocation to be highly organized in liquid-ordered plasma membrane nanodomains, the core process underlying this unusual pathway of protein secretion.

Disulfiram inhibits IL-1ß secretion and inflammatory cells recruitment in Aspergillus fumigatus keratitis.

PURPOSE: Disulfiram, an inhibitor of gasdermin D-induced pore formation, is known to suppress interleukin (IL)-1ß secretion and pyroptosis. However, its effects on fungal keratitis remain unknown. Therefore, we investigated the role of disulfiram in Aspergillus fumigatus keratitis. METHODS: In vitro, Cell Count Kit-8 (CCK8) assay and cell scratch test were performed to determine optimal concentration. In vivo and in vitro experiments were conducted in a mouse model, human neutrophils, and mouse peritoneal macrophages. We pre-treated the mice or cells with disulfiram and infected them with A. fumigatus at specific times. We subsequently evaluated the development of fungal keratitis lesions, the recruitment of inflammatory cells, and the production of inflammatory cytokines using slit lamp microscopy, clinical evaluation, quantitative reverse transcription polymerase chain reaction, immunofluorescence staining, enzyme-linked immunosorbent assay, and western blotting. We also used slit lamp microscopy and clinical evaluation to assess the effect of natamycin with or without disulfiram. RESULTS: Disulfiram at 20 µM has no significant cytotoxic effect and does not affect cell migration. In the mouse model, disulfiram significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and down-regulated myeloperoxidase and nitric oxide synthase levels at earlier stages of infection. Disulfiram had no effect on IL-1ß production and maturation, but it inhibited IL-1ß secretion in macrophages. Disulfiram combined with natamycin significantly increased corneal transparency in the mice model. CONCLUSION: Overall, disulfiram reduced the host immune response in fungal keratitis by attenuating neutrophil and macrophage recruitment and inhibiting IL-1ß secretion in macrophages. Disulfiram in combination with antifungal agents may serve as a novel therapeutic method for reducing corneal opacity in fungal keratitis.

Measuring Voltage-Current Characteristics of Tethered Bilayer Lipid Membranes to Determine the Electro-Insertion Properties of Analytes.

Tethered bilayer lipid membranes (tBLMs) anchored to a solid substrate can be prepared and individual triangular voltage ramps from zero to 500 mV with a period of 2-10 ms applied to give membrane voltage dependencies with and without the addition of drugs and analytes in order to measure their electro-insertion properties.

Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs).

Model lipid bilayers tethered to a gold substrate with molecular tethers are constructed. The conductance versus temperature dependence curve is then obtained. Here, a method to measure the activation energy for translocation of an ion through existing transmembrane pores in a sparsely tethered bilayer lipid membranes is presented.

Anticorrelated position fluctuation of lipids in forming membrane water pores: molecular dynamics simulations study with dengue virus capsid protein.

The traffic of molecules into or out of cells is regulated by many membrane-associated mechanisms. Membrane pores are considered as one of the major passage mechanisms, although molecular-level understanding of pore formation is still vague. The opening of a membrane pore depends on many factors, including the influence of some proteins. The ability of the cell-penetrating peptides and supercharged proteins to form membrane pores has been reported. We studied pore formation through dipalmitoylphosphatidylcholine (DPPC) lipid bilayers by supercharged dengue virus capsid (C) protein. Atomistic molecular dynamics simulations confirmed the formation of membrane pores by a combined effect of the C protein and the membrane electric field. Analyses of simulated trajectories showed highly correlated vertical position fluctuations between the Cα atom of the membrane-anchored arginine residues and the phosphorus atoms of the surrounding DPPC lipids. Certain regions of the bilayer were negatively correlated while the others were positively correlated with respect to the fluctuations of the Cα atom of the anchored arginine residues. When positively correlated lipids in one leaflet vertically aligned with the negatively correlated lipids in the other leaflet, a local anticorrelated region was generated by weakening the bilayer. The membrane pore was always formed close to this anticorrelated region. Once formed, the C protein followed the hydrated pathway provided by the water-filled pores to cross the membrane.Communicated by Ramaswamy H. Sarma.

Delivery of Ultrasmall Nanoparticles to the Cytosolic Compartment of Pyroptotic J774A.1 Macrophages via GSDMDNterm Membrane Pores.

Endosome capture is a major physiological barrier to the successful delivery of nanomedicine. Here, we found a strategy to deliver ultrasmall nanoparticles (<10 nm) to the cytosolic compartment of pyroptotic cells with spontaneous endosomal escape. To mimic pathological pyroptotic cells, J774A.1 macrophages were stimulated with lipopolysaccharide (LPS) plus nigericin (Nig) or adenosine triphosphate (ATP) to form specific gasdermin D protein-driven membrane pores at an N-terminal domain (GSDMDNterm). Through GSDMDNterm membrane pores, both anionic and cationic nanoparticles (NPs) with diameters less than 10 nm were accessed into the cytosolic compartment of pyroptotic cells in an energy- and receptor-independent manner, while NPs larger than the size of GSDMDNterm membrane pores failed to enter pyroptotic cells. NPs pass through GSDMDNterm membrane pores via free diffusion and then access into the cytoplasm of pyroptotic cells in a microtubule-independent manner. Interestingly, we found that LPS-primed NPs may act as Trojan horse, deliver extracellular LPS into normal cells through endocytosis, and in turn induce GSDMDNterm membrane pores, which facilitate further internalization of NPs. This study presented a straightforward method of distinguishing normal and pyroptotic cells through GSDMD membrane pores, implicating their potential application in monitoring the delivery of desired nanomedicines in pyroptosis-related diseases and conditions.

Repairing plasma membrane damage in regulated necrotic cell death.

The plasma membrane performs a central role in maintaining cellular homeostasis and viability by acting as a semi-permeable barrier separating the cell from its surroundings. Under physiological conditions, it is constantly exposed to different kinds of stress, such as from pore-forming proteins/toxins and mechanical activity, that compromises its integrity resulting in cells developing various ways to cope with these dangers to survive. These plasma membrane repair mechanisms are initiated by the rapid influx of extracellular Ca2+ ions and are thus hinged on the activity of various Ca2+-binding proteins. The cell's response to membrane damage also depends on the nature and extent of the stimuli as well as the cell type, and the mechanisms involved are believed to be not mutually exclusive. In regulated necrotic cell death, specifically necroptosis, pyroptosis, and ferroptosis, plasma membrane damage ultimately causes cell lysis and the release of immunomodulating damage-associated molecular patterns. Here, I will discuss how these three cell death pathways are counterbalanced by the action of ESCRT (Endosomal Sorting Complex Required for Transport)-III-dependent plasma membrane repair mechanism, that eventually affects the profile of released cytokines and cell-to-cell communication. These highlight a crucial role that plasma membrane repair play in regulated necrosis, and its potential as a viable target to modulate the immune responses associated with these pathways in the context of the various human pathologies where these cell death modalities are implicated.

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Pore formation in regulated cell death.

The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.

Ultrastructural Characteristics of DHA-Induced Pyroptosis.

Microglial cells are resident macrophages of the central nervous system (CNS) that respond to bioactive lipids such as docosahexaenoic acid (DHA). Low micromolar concentrations of DHA typically promote anti-inflammatory functions of microglia, but higher concentrations result in a form of pro-inflammatory programmed cell death known as pyroptosis. This study used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to investigate the morphological characteristics of pyroptosis in BV-2 microglial cells following exposure to 200 µM DHA. Vehicle-treated cells are characterized by extended processes, spine-like projections or 0.4 to 5.2 µm in length, and numerous extracellular vesicles (EVs) tethered to the surface of the plasma membrane. In contrast to vehicle-treated cells, gross abnormalities are observed after treating cells with 200 µM DHA for 4 h. These include the appearance of numerous pits or pores of varying sizes across the cell surface, structural collapse and flattening of the cell shape. Moreover, EVs and spines were lost following DHA treatment, possibly due to release from the cell surface. The membrane pores appear after DHA treatment initially measured ~ 30 nm, consistent with the previously reported gasdermin D (GSDMD) pore complexes. Complete collapse of cytoplasmic organization and loss of nuclear envelope integrity were also observed in DHA-treated cells. These processes are morphologically distinct from the changes that occur during cisplatin-induced apoptosis, such as the appearance of apoptotic bodies and tightly packed organelles, and the maintenance of EVs and nuclear envelope integrity. Cumulatively, this study provides a systematic description of the ultrastructural characteristics of DHA-induced pyroptosis, including distinguishing features that differentiate this process from apoptosis.

Nanopore Passport Control for Substrate-Specific Translocation.

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substrate-specific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

Molecular dynamics study of membrane permeabilization by wild-type and mutant lytic peptides from the non-enveloped Flock House virus.

Flock House virus (FHV) serves as a model system for understanding infection mechanisms utilized by non-enveloped viruses to transport across cellular membranes. During the infection cycle of FHV, a fundamental stage involves disruption of the endosomal membrane by membrane active peptides, following externalization of the peptides from the capsid interior. The FHV lytic agents are the 44 C-terminal amino acids residues of the capsid protein, which are auto-catalytically cleaved during the capsid maturation process. The cleaved peptides are termed γ peptides. In this study, we perform multi-scale molecular dynamics simulations including 40 µs all-atom molecular dynamics simulations to study the behavior of pre-inserted transmembrane lytic peptides at a high concentration in a neutral membrane. We study the dynamical organization among peptides to form oligomeric bundles in four systems including the wild-type γ peptide and three mutant forms; namely, a truncation mutant in which the 23 C-terminal residues are deleted (γ1), a construct where the 8 C-terminal residues of γ are fused to γ1 (Δ385-399 γ) and a single-point mutant (F402A γ), all of which have been experimentally shown to drastically affect infectivity and lytic activity compared to the wild-type γ. Our results shed light on the actions of varied forms of the FHV lytic peptide including membrane insertion, trans-membrane stability, peptide oligomerization, water permeation activity and dynamic pore formation. Findings from this study provide detailed structural information and rationale for the differences in lytic activity among variants of FHV γ.

Colicin U from Shigella boydii Forms Voltage-Dependent Pores.

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.