Modulation of Voltage-Gating and Hysteresis of Lysenin Channels by Cu2+ Ions.

The intricate voltage regulation presented by lysenin channels reconstituted in artificial lipid membranes leads to a strong hysteresis in conductance, bistability, and memory. Prior investigations on lysenin channels indicate that the hysteresis is modulated by multivalent cations which are also capable of eliciting single-step conformational changes and transitions to stable closed or sub-conducting states. However, the influence on voltage regulation of Cu2+ ions, capable of completely closing the lysenin channels in a two-step process, was not sufficiently addressed. In this respect, we employed electrophysiology approaches to investigate the response of lysenin channels to variable voltage stimuli in the presence of small concentrations of Cu2+ ions. Our experimental results showed that the hysteretic behavior, recorded in response to variable voltage ramps, is accentuated in the presence of Cu2+ ions. Using simultaneous AC/DC stimulation, we were able to determine that Cu2+ prevents the reopening of channels previously closed by depolarizing potentials and the channels remain in the closed state even in the absence of a transmembrane voltage. In addition, we showed that Cu2+ addition reinstates the voltage gating and hysteretic behavior of lysenin channels reconstituted in neutral lipid membranes in which lysenin channels lose their voltage-regulating properties. In the presence of Cu2+ ions, lysenin not only regained the voltage gating but also behaved like a long-term molecular memory controlled by electrical potentials.

Hypo-Osmotic Stress and Pore-Forming Toxins Adjust the Lipid Order in Sheep Red Blood Cell Membranes.

Lipid ordering in cell membranes has been increasingly recognized as an important factor in establishing and regulating a large variety of biological functions. Multiple investigations into lipid organization focused on assessing ordering from temperature-induced phase transitions, which are often well outside the physiological range. However, particular stresses elicited by environmental factors, such as hypo-osmotic stress or protein insertion into membranes, with respect to changes in lipid status and ordering at constant temperature are insufficiently described. To fill these gaps in our knowledge, we exploited the well-established ability of environmentally sensitive membrane probes to detect intramembrane changes at the molecular level. Our steady state fluorescence spectroscopy experiments focused on assessing changes in optical responses of Laurdan and diphenylhexatriene upon exposure of red blood cells to hypo-osmotic stress and pore-forming toxins at room temperature. We verified our utilized experimental systems by a direct comparison of the results with prior reports on artificial membranes and cholesterol-depleted membranes undergoing temperature changes. The significant changes observed in the lipid order after exposure to hypo-osmotic stress or pore-forming toxins resembled phase transitions of lipids in membranes, which we explained by considering the short-range interactions between membrane components and the hydrophobic mismatch between membrane thickness and inserted proteins. Our results suggest that measurements of optical responses from the membrane probes constitute an appropriate method for assessing the status of lipids and phase transitions in target membranes exposed to mechanical stresses or upon the insertion of transmembrane proteins.

Double-stemmed and split structural variants of fluorescent RNA Mango aptamers.

Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is ∼75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rational design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are cotranscribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA-RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Experimental Investigations on the Conductance of Lipid Membranes under Differential Hydrostatic Pressure.

The unassisted transport of inorganic ions through lipid membranes has become increasingly relevant to an expansive range of biological phenomena. Recent simulations indicate a strong influence of a lipid membrane's curvature on its permeability, which may be part of the overall cell sensitivity to mechanical stimulation. However, most ionic permeability experiments employ a flat, uncurved lipid membrane, which disregards the physiological relevance of curvature on such investigations. To fill this gap in our knowledge, we adapted a traditional experimental system consisting of a planar lipid membrane, which we exposed to a controlled, differential hydrostatic pressure. Our electrophysiology experiments indicate a strong correlation between the changes in membrane geometry elicited by the application of pressure, as inferred from capacitance measurements, and the resulting conductance. Our experiments also confirmed the well-established influence of cholesterol addition to lipid membranes in adjusting their mechanical properties and overall permeability. Therefore, the proposed experimental system may prove useful for a better understanding of the intricate connections between membrane mechanics and adjustments of cellular functionalities upon mechanical stimulation, as well as for confirmation of predictions made by simulations and theoretical modeling.

The Ionic Selectivity of Lysenin Channels in Open and Sub-Conducting States.

The electrochemical gradients established across cell membranes are paramount for the execution of biological functions. Besides ion channels, other transporters, such as exogenous pore-forming toxins, may present ionic selectivity upon reconstitution in natural and artificial lipid membranes and contribute to the electrochemical gradients. In this context, we utilized electrophysiology approaches to assess the ionic selectivity of the pore-forming toxin lysenin reconstituted in planar bilayer lipid membranes. The membrane voltages were determined from the reversal potentials recorded upon channel exposure to asymmetrical ionic conditions, and the permeability ratios were calculated from the fit with the Goldman-Hodgkin-Katz equation. Our work shows that lysenin channels are ion-selective and the determined permeability coefficients are cation and anion-species dependent. We also exploited the unique property of lysenin channels to transition to a stable sub-conducting state upon exposure to calcium ions and assessed their subsequent change in ionic selectivity. The observed loss of selectivity was implemented in an electrical model describing the dependency of reversal potentials on calcium concentration. In conclusion, our work demonstrates that this pore-forming toxin presents ionic selectivity but this is adjusted by the particular conduction state of the channels.

Rapid Production and Purification of Dye-Loaded Liposomes by Electrodialysis-Driven Depletion.

Liposomes are spherical-shaped vesicles that enclose an aqueous milieu surrounded by bilayer or multilayer membranes formed by self-assembly of lipid molecules. They are intensively exploited as either model membranes for fundamental studies or as vehicles for delivery of active substances in vivo and in vitro. Irrespective of the method adopted for production of loaded liposomes, obtaining the final purified product is often achieved by employing multiple, time consuming steps. To alleviate this problem, we propose a simplified approach for concomitant production and purification of loaded liposomes by exploiting the Electrodialysis-Driven Depletion of charged molecules from solutions. Our investigations show that electrically-driven migration of charged detergent and dye molecules from solutions that include natural or synthetic lipid mixtures leads to rapid self-assembly of loaded, purified liposomes, as inferred from microscopy and fluorescence spectroscopy assessments. In addition, the same procedure was successfully applied for incorporating PEGylated lipids into the membranes for the purpose of enabling long-circulation times needed for potential in vivo applications. Dynamic Light Scattering analyses and comparison of electrically-formed liposomes with liposomes produced by sonication or extrusion suggest potential use for numerous in vitro and in vivo applications.

Liposomes Prevent In Vitro Hemolysis Induced by Streptolysin O and Lysenin.

The need for alternatives to antibiotics in the fight against infectious diseases has inspired scientists to focus on antivirulence factors instead of the microorganisms themselves. In this respect, prior work indicates that tiny, enclosed bilayer lipid membranes (liposomes) have the potential to compete with cellular targets for toxin binding, hence preventing their biological attack and aiding with their clearance. The effectiveness of liposomes as decoy targets depends on their availability in the host and how rapidly they are cleared from the circulation. Although liposome PEGylation may improve their circulation time, little is known about how such a modification influences their interactions with antivirulence factors. To fill this gap in knowledge, we investigated regular and long-circulating liposomes for their ability to prevent in vitro red blood cell hemolysis induced by two potent lytic toxins, lysenin and streptolysin O. Our explorations indicate that both regular and long-circulating liposomes are capable of similarly preventing lysis induced by streptolysin O. In contrast, PEGylation reduced the effectiveness against lysenin-induced hemolysis and altered binding dynamics. These results suggest that toxin removal by long-circulating liposomes is feasible, yet dependent on the particular virulence factor under scrutiny.

Lysenin Channels as Sensors for Ions and Molecules.

Lysenin is a pore-forming protein extracted from the earthworm Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications.

Kinetic Exclusion Assay of Biomolecules by Aptamer Capture.

DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features recommend aptamers as candidates for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, we exploited the analytical capabilities offered by the kinetic exclusion assay technology to measure the affinity of fluorescent aptamers for their thrombin target and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective Kd of the binding in the sub-nanomolar range. However, our experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct Kd populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine "unknown" concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM-nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements.

Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin.

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin's transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel's conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Center of Biomedical Research Excellence in Matrix Biology: Building Research Infrastructure, Supporting Young Researchers, and Fostering Collaboration.

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

An Effective Electric Dipole Model for Voltage-induced Gating Mechanism of Lysenin.

Lysenin is a pore-forming toxin, which self-inserts open channels into sphingomyelin containing membranes and is known to be voltage regulated. The mechanistic details of its voltage gating mechanism, however, remains elusive despite much recent efforts. Here, we have employed a novel combination of experimental and computational techniques to examine a model for voltage gating, that is based on the existence of an "effective electric dipole" inspired by recent reported structures of lysenin. We support this mechanism by the observations that (i) the charge-reversal and neutralization substitutions in lysenin result in changing its electrical gating properties by modifying the strength of the dipole, and (ii) an increase in the viscosity of the solvent increases the drag force and slows down the gating. In addition, our molecular dynamics (MD) simulations of membrane-embedded lysenin provide a mechanistic picture for lysenin conformational changes, which reveals, for the first time, the existence of a lipid-dependent bulge region in the pore-forming module of lysenin, which may explain the gating mechanism of lysenin at a molecular level.

Defect Engineering of ZnO Nanoparticles for Bioimaging Applications.

Many promising attributes of ZnO nanoparticles (nZnO) have led to their utilization in numerous electronic devices and biomedical technologies. nZnO fabrication methods can create a variety of intrinsic defects that modulate the properties of nZnO, which can be exploited for various purposes. Here we developed a new synthesis procedure that controls certain defects in pure nZnO that are theorized to contribute to the n-type conductivity of the material. Interestingly, this procedure created defects that reduced the nanoparticle band gap to ∼3.1 eV and generated strong emissions in the violet to blue region while minimizing the defects responsible for the more commonly observed broad green emissions. Several characterization techniques including thermogravimetric analysis, Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, transmission electron microscopy, Raman, photoluminescence, and inductively coupled plasma mass spectrometry were employed to verify the sample purity, assess how modifications in the synthesis procedure affect the various defects states, and understand how these alterations impact the physical properties. Since the band gap significantly decreased and a relatively narrow visible emissions band was created by these defects, we investigated utilizing these new nZnO for bioimaging applications using traditional fluorescent microscopy techniques. Although most nZnO generally require UV excitation sources to produce emissions, we demonstrate that reducing the band gap allows for a 405 nm laser to sufficiently excite the nanoparticles to detect their emissions during live-cell imaging experiments using a confocal microscope. This work lays the foundation for the use of these new nZnO in various bioimaging applications and enables researchers to investigate the interactions of pure nZnO with cells through fluorescence-based imaging techniques.

Insights into the Voltage Regulation Mechanism of the Pore-Forming Toxin Lysenin.

Lysenin, a pore forming toxin (PFT) extracted from Eisenia fetida, inserts voltage-regulated channels into artificial lipid membranes containing sphingomyelin. The voltage-induced gating leads to a strong static hysteresis in conductance, which endows lysenin with molecular memory capabilities. To explain this history-dependent behavior, we hypothesized a gating mechanism that implies the movement of a voltage domain sensor from an aqueous environment into the hydrophobic core of the membrane under the influence of an external electric field. In this work, we employed electrophysiology approaches to investigate the effects of ionic screening elicited by metal cations on the voltage-induced gating and hysteresis in conductance of lysenin channels exposed to oscillatory voltage stimuli. Our experimental data show that screening of the voltage sensor domain strongly affects the voltage regulation only during inactivation (channel closing). In contrast, channel reactivation (reopening) presents a more stable, almost invariant voltage dependency. Additionally, in the presence of anionic Adenosine 5'-triphosphate (ATP), which binds at a different site in the channel's structure and occludes the conducting pathway, both inactivation and reactivation pathways are significantly affected. Therefore, the movement of the voltage domain sensor into a physically different environment that precludes electrostatically bound ions may be an integral part of the gating mechanism.

ZnO nanoparticles modulate the ionic transport and voltage regulation of lysenin nanochannels.

BACKGROUND: The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate. RESULTS: The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO2 NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels. CONCLUSION: We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization.

Stochastic sensing of Angiotensin II with lysenin channels.

The ability of pore-forming proteins to interact with various analytes has found vast applicability in single molecule sensing and characterization. In spite of their abundance in organisms from all kingdoms of life, only a few pore-forming proteins have been successfully reconstituted in artificial membrane systems for sensing purposes. Lysenin, a pore-forming toxin extracted from the earthworm E. fetida, inserts large conductance nanopores in lipid membranes containing sphingomyelin. Here we show that single lysenin channels may function as stochastic nanosensors by allowing the short cationic peptide angiotensin II to be electrophoretically driven through the conducting pathway. Long-term translocation experiments performed using large populations of lysenin channels allowed unequivocal identification of the unmodified analyte by Liquid Chromatography-Mass Spectrometry. However, application of reverse voltages or irreversible blockage of the macroscopic conductance of lysenin channels by chitosan addition prevented analyte translocation. This investigation demonstrates that lysenin channels have the potential to function as nano-sensing devices capable of single peptide molecule identification and characterization, which may be further extended to other macromolecular analytes.

Modeling and Analysis of Intercalant Effects on Circular DNA Conformation.

The large-scale conformation of DNA molecules plays a critical role in many basic elements of cellular functionality and viability. By targeting the structural properties of DNA, many cancer drugs, such as anthracyclines, effectively inhibit tumor growth but can also produce dangerous side effects. To enhance the development of innovative medications, rapid screening of structural changes to DNA can provide important insight into their mechanism of interaction. In this study, we report changes to circular DNA conformation from intercalation with ethidium bromide using all-atom molecular dynamics simulations and characterized experimentally by translocation through a silicon nitride solid-state nanopore. Our measurements reveal three distinct current blockade levels and a 6-fold increase in translocation times for ethidium bromide-treated circular DNA as compared to untreated circular DNA. We attribute these increases to changes in the supercoiled configuration hypothesized to be branched or looped structures formed in the circular DNA molecule. Further evidence of the conformational changes is demonstrated by qualitative atomic force microscopy analysis. These results expand the current methodology for predicting and characterizing DNA tertiary structure and advance nanopore technology as a platform for deciphering structural changes of other important biomolecules.

Purinergic control of lysenin's transport and voltage-gating properties.

Lysenin, a pore-forming protein extracted from the coelomic fluid of the earthworm Eisenia foetida, manifests cytolytic activity by inserting large conductance pores in host membranes containing sphingomyelin. In the present study, we found that adenosine phosphates control the biological activity of lysenin channels inserted into planar lipid membranes with respect to their macroscopic conductance and voltage-induced gating. Addition of ATP, ADP, or AMP decreased the macroscopic conductance of lysenin channels in a concentration-dependent manner, with ATP being the most potent inhibitor and AMP the least. ATP removal from the bulk solutions by buffer exchange quickly reinstated the macroscopic conductance and demonstrated reversibility. Single-channel experiments pointed to an inhibition mechanism that most probably relies on electrostatic binding and partial occlusion of the channel-conducting pathway, rather than ligand gating induced by the highly charged phosphates. The Hill analysis of the changes in macroscopic conduction as a function of the inhibitor concentration suggested cooperative binding as descriptive of the inhibition process. Ionic screening significantly reduced the ATP inhibitory efficacy, in support of the electrostatic binding hypothesis. In addition to conductance modulation, purinergic control over the biological activity of lysenin channels has also been observed to manifest as changes of the voltage-induced gating profile. Our analysis strongly suggests that not only the inhibitor's charge but also its ability to adopt a folded conformation may explain the differences in the observed influence of ATP, ADP, and AMP on lysenin's biological activity.

Intramembrane congestion effects on lysenin channel voltage-induced gating.

All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin's preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease.