Bilayer Model Membranes Research Articles

Aqueous Ionic Liquid Mixtures as Minimal Models of Lipid Bilayer Membranes.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, "healthy" charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich "beads" clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.

Concentration-Dependent Effects of Curcumin on Membrane Permeability and Structure.

Growing evidence suggests that many bioactive molecules can nonspecifically modulate the physicochemical properties of membranes and influence the action of embedded membrane proteins. This study investigates the interactions of curcumin with protein-free model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC with cholesterol (4/1 mol ratio). The focus is on the capability of curcumin to modify membrane barrier properties such as water permeability assayed through the droplet interface bilayer (DIB) model membrane. For pure DOPC, our findings show a concentration-dependent biphasic effect: a reduction in water permeability is observed at low concentrations (up to 2 mol %), whereas at high concentrations of curcumin, water permeability increases. In the presence of cholesterol, we observed an overall reduction in water permeability. A combination of complementary experimental methods, including phase transition parameters studied by differential scanning calorimetry (DSC) and structural properties measured by attenuated total reflectance (ATR)-FTIR, provides a deeper understanding of concentration-dependent interactions of curcumin with DOPC bilayers in the absence and presence of cholesterol. Our experimental findings align with a molecular mechanism of curcumin's interaction with model membranes, wherein its effect is contingent on its concentration. At low concentrations, curcumin binds to the lipid-water interface through hydrogen bonding with the phosphate headgroup, thereby obstructing the transport of water molecules. Conversely, at high concentrations, curcumin permeates the acyl chain region, inducing packing disorders and demonstrating evidence of phase separation. Enhanced knowledge of the impact of curcumin on membranes, which, in turn, can affect protein function, is likely to be beneficial for the successful translation of curcumin into effective medicine.

Insight into the effect of ibuprofen on the permeability of the membrane: a molecular dynamic simulation study

Studying interactions between drugs and cell membranes is of great interest to designing novel drugs, optimizing drug delivery, and discerning drug mechanism action. In this study, we investigated the physical properties of the bilayer membrane model of POPC upon interaction with ibuprofen (IBU) using molecular dynamics simulations. The area per lipid (APL) was calculated to describe the effect of ibuprofen on the packing properties of the lipid bilayer. The APL was 0.58 nm2 and 0.63 nm2 for the membrane in low and high IBU respectively, and 0.57 nm2 for the membrane without IBU. Our finding showed that the mean square deviation (MSD) increased with increased ibuprofen content. In addition, the order parameter for the hydrocarbon chain of lipids increased with increased ibuprofen content. There was an increment in the transfer free energy after the head group region while it was maximum in the hydrophobic core for hydrogen peroxide (H2O2) (∼6.2 kcal.mol−1) and H2O (∼3.4 kcal.mol−1) which then decreased to respective values of (∼4.6 kcal.mol−1), and (∼2.3 kcal.mol−1) at the center of the bilayer in the presence of IBU. It seems that in the presence of ibuprofen, the free energy profile of the permeability of water and H2O2 significantly decreased. These findings show that ibuprofen significantly influences the physical properties of the bilayer by decreasing the packing and intermolecular interaction in the hydrocarbon chain region and increasing the water permeability of the bilayer. These results may provide insights into the local cytotoxic side effects of ibuprofen and its underlying molecular mechanisms. Communicated by Ramaswamy H. Sarma

Absolute scattering length density profile of liposome bilayers obtained by SAXS combined with GIXOS: a tool to determine model biomembrane structure

Lipid membranes play an essential role in biology, acting as host matrices for biomolecules like proteins and facilitating their functions. Their structures and structural responses to physiologically relevant interactions (i.e. with membrane proteins) provide key information for understanding biophysical mechanisms. Hence, there is a crucial need of methods to understand the effects of membrane host molecules on the lipid bilayer structure. Here, a purely experimental method is presented for obtaining the absolute scattering length density profile and the area per lipid of liposomal bilayers, by aiding the analysis of small-angle X-ray scattering (SAXS) data with the volume of bare headgroups obtained from grazing-incidence X-ray off-specular scattering (GIXOS) data of monolayers of the same model membrane lipid composition. The GIXOS data experimentally demonstrate that the variation of the bare headgroup volume upon change in lipid packing density is small enough to allow its usage as a reference value without knowing the lipid packing stage in a bilayer. This approach also has the advantage that the reference volume is obtained in the same aqueous environment as used for the model membrane bilayers. The validity of this method is demonstrated using several typical membrane compositions, as well as one example of a phospholipid membrane with an incorporated transmembrane peptide. This methodology allows us to obtain absolute scale rather than relative scale values using solely X-ray-based instrumentation, retaining a similar resolution to SAXS experiments. The method presented has high potential for understanding the structural effects of membrane proteins on the biomembrane structure.

The Effect of Benzyl Alcohol on the Voltage-Current Characteristics of Tethered Lipid Bilayers.

In this study a lipid bilayer membrane model was used in which the bilayer is tethered to a solid substrate with molecular tethers. Voltage-current (V-I) measurements of the tethered bilayer membranes (tBLM) and tBLM with benzyl alcohol (BZA) incorporated in their structures, were measured using triangular voltage ramps of 0-500mV. The temperature dependence of the conductance deduced from the V-I measurements are described. An evaluation of the activation energies for electrical conductance showed that BZA decreased the activation/ Born energies for ionic conduction of tethered lipid membranes. It is concluded that BZA increased the average pore radius of the tBLM.

Structural Phenomena in a Vesicle Membrane Obtained through an Evolution Experiment: A Study Based on MD Simulations.

The chemical evolution of biomolecules was clearly affected by the overall extreme environmental conditions found on Early Earth. Periodic temperature changes inside the Earth's crust may have played a role in the emergence and survival of functional peptides embedded in vesicular compartments. In this study, all-atom molecular dynamic (MD) simulations were used to elucidate the effect of temperature on the properties of functionalized vesicle membranes. A plausible prebiotic system was selected, constituted by a model membrane bilayer from an equimolar mixture of long-chain fatty acids and fatty amines, and an octapeptide, KSPFPFAA, previously identified as an optimized functional peptide in an evolution experiment. This peptide tends to form the largest spontaneous aggregates at higher temperatures, thereby enhancing the pore-formation process and the eventual transfer of essential molecules in a prebiotic scenario. The analyses also suggest that peptide-amphiphile interactions affect the structural properties of the membrane, with a significant increase in the degree of interdigitation at the lowest temperatures under study.

Exploring the mechanism of photosensitizer conjugation on membrane perturbation of antimicrobial peptide: A multiscale molecular simulation study

Antimicrobial peptides (AMPs) exert their biological functions by perturbation with cellular membrane. Conjugation of AMPs with photosensitizer (PS) is a promising strategy for enhancing the efficacy and reducing systemic toxicity of AMPs. However, it is still elusive how the conjugated PS impacts the perturbation of AMPs on cell membrane from molecular level. Here, we addressed this issue by a multiscale computational strategy on pyropheophorbide-a (PPA) conjugated K6L9 (PPA-K6L9), a PS-AMP conjugate developed by us previously. Our atomistic molecular dynamics (MD) simulations revealed that the porphyrin moiety of PPA enhanced the stability of the conjugate in a lipid bilayer membrane model. Moreover, such moiety also maintained the amphipathic structure of K6L9, which is crucial for membrane pore formation. Coarse-grained MD simulations further showed that the conjugates aggregated in membrane environment and formed more stable toroidal pores with respect to K6L9 alone, suggesting the conjugation of PPA may enhance the membrane-disruption activity of K6L9. Consistent with this, our cellular experiments confirmed that PPA-K6L9 was more toxic to 4 T1 tumor cells than K6L9. This study provides insights into the mechanism by which PS-AMP conjugates disrupt cellular membranes and could aid in the design of more potent AMP conjugates.

Cholesterol solubility in mixed DMPE/DMPC bilayers as determined by small angle X-ray scattering

Small angle X-ray scattering measurements under ambient conditions (T ≈ 294 K) provide evidence for the formation of separate domains in a ternary, mixed phospholipid ([DMPE]/[DMPC] = 3/1) / cholesterol model bilayer membrane. As we interpret these results, the domains contain cholesterol and DMPC, with which cholesterol is known to preferentially interact in a binary model membrane (solubility limit, mol fraction cholesterol 0.5), as compared to DMPE (solubility limit, mol fraction cholesterol 0.45). The solubility limit for the ternary system is mol fraction cholesterol 0.2–0.3. Although literature EPR spectra find that non-crystalline, cholesterol bilayer domains may be present even prior to the observation of cholesterol crystal diffraction, X-ray scattering cannot detect their presence.

The role of hydrophobic patches of de novo designed MSI-78 and VG16KRKP antimicrobial peptides on fragmenting model bilayer membranes

Antimicrobial peptides (AMPs) with cell membrane lysing capability are considered potential candidates for the development of the next generation of antibiotics. Designing novel AMPs requires an in-depth understanding of the mechanism of action of the peptides. In this work, we used various biophysical techniques including 31P solid-state NMR to examine the interaction of model membranes with amphipathic de novo-designed peptides. Two such peptides, MSI-78 and VG16KRKP, were designed with different hydrophobicity and positive charges. The model lipid membranes were constituted by mixing lipids of varying degrees of ‘area per lipid’ (APL), which directly affected the packing properties of the membrane. The observed emergence of the isotropic peak in 31P NMR spectra as a function of time is a consequence of the fragmentation of the membrane mediated by the peptide interaction. The factors such as the charges, overall hydrophilicity of the AMPs, as well as lipid membrane packing, contributed to the kinetics of membrane fragmentation. Furthermore, we anticipate the designed AMPs follow the carpet and toroidal pore mechanisms when lysing the cell membrane. This study highlights the significance of the effect of the overall charges and the hydrophobicity of the novel AMPs designed for antimicrobial activity.

Thermal-controlled cellular uptake of "hot" nanoparticles.

Nanoparticles (NPs) have shown immense potential in the field of biomedical applications, particularly in NP-based photothermal therapy, which offers a remote-controlled approach to achieve precise temperature control for site-specific heating and sub-cellular tumor treatment. However, the molecular mechanisms underlying related cellular activities, such as the cellular uptake behavior of irradiated NPs in photothermal effects, remain elusive. In this study, we conducted a thorough investigation of the interaction between an irradiated NP with elevated temperature (ranging from 270 to 360 K) and a model bilayer membrane composed of DPPC or DOPC using nonequilibrium coarse-grained molecular dynamics simulations with the implicit-solvent Dry Martini force field. We observe that the interaction between a "hot" NP and a membrane is thermally regulated. In addition, the wrapping of membranes around NPs exhibits a strong dependence on the temperature of the irradiated NP, demonstrating a step-like change in behavior. This membrane wrapping effect is attributed to the heat conduction between NPs and membrane lipids, which occurs almost simultaneously with the membrane deformation and wrapping of NPs during the NP-membrane interaction process. Especially, during the process of heat conduction, a gel-to-fluid phase transition of the membrane may occur, which plays a crucial role in determining the deformation behavior of the membrane. Moreover, it is found that the membrane lipids in the two leaflets exhibit obvious and asymmetric molecular-level responses to heat flux, characterized by significant changes in packing states (e.g., the order parameter of lipid tails and area per lipid) and possible interdigitation between lipids. Furthermore, the thermal-controlled wrapping effect is tightly linked to the properties of NPs (e.g., size, NP-lipid affinity) and lipid species. Our findings are valuable for comprehending the thermal-regulated cellular internalization of NPs and offer insights into devising strategies to precisely modulate NP endocytosis by exploiting the interplay between heating and NP properties.

Molecular interactions of hesperidin with DMPC/cholesterol bilayers

Since cell membranes are complex systems, the use of model lipid bilayers is quite important for the study of their interactions with bioactive molecules. Mammalian cell membranes require cholesterol (CHOL) for their structure and function. For this reason, the mixtures of phospholipid and cholesterol are necessary to use in model membrane studies to better simulate the real systems. In the present study, we investigated the effect of the incorporation of hesperidin in model membranes consisting of dimyristoylphosphatidylcholine (DMPC) and CHOL by using differential scanning calorimetry (DSC), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and atomic force microscopy (AFM). ATR-FTIR results demonstrated that hesperidin increases the fluidity of the DMPC/CHOL binary system. DSC findings indicated that the presence of 5 mol% hesperidin induces a broadening of the main phase transition consisting of three overlapping components. AFM experiments showed that hesperidin increases the thickness of DMPC/CHOL lipid bilayer model membranes. In addition to experimental results, molecular docking studies were conducted with hesperidin and human lanosterol synthase (LS), which is an enzyme found in the final step of cholesterol synthesis, to characterize hesperidin's interactions with its surrounding via its hydroxyl and oxygen groups. Then, hesperidin's ADME/Tox (absorption, distribution, metabolism, excretion and toxicity) profile was computed to see the potential impact on living system. In conclusion, considering the data obtained from experimental studies, this work ensures molecular insights in the interaction between a flavonoid, as an antioxidant drug model, and lipids mimicking those found in mammalian membranes. Moreover, computational studies demonstrated that hesperidin may be a great potential for use as a therapeutic agent for hypercholesterolemia due to its antioxidant property.

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.

Assessment of a host-guest interaction in a bilayer membrane model.

Supramolecular interactions are well recognized and many of them have been extensively studied in chemistry. The formation of supramolecular complexes that rely on weak force interactions are less well studied in bilayer membranes. Herein, a supported bilayer membrane is used to probe the penetration of a complex between tetracycline and a macrocyclic polyether. In a number of bacterial systems, the presence of the macrocycle has been found to significantly enhance the potency of the antimicrobial in vitro. The crown·tetracycline complex has been characterized in solution, neutron reflectometry has probed complex penetration, and the phenomena have been modeled by computational methods.

Challenges and Methods for the Study of CPP Translocation Mechanisms.

Fluorescence-based methods are widely used to detect crossing of peptides across model or biological membranes. For membrane-active peptides, i.e., peptides that have strong membrane tropism, fluorescence experiments must be accompanied by relevant controls, otherwise they can lead to inconsistent interpretation and underestimation of their limitations. Here we describe how to prepare samples to study fluorescent peptide crossing droplet interface bilayer (model membrane) or cell membrane (biological membrane) and the pitfalls that can affect observational qualitative and quantitative data.

Identification of a critical lipid ratio in raft-like phases exposed to nitric oxide: An AFM study

Lipid rafts are discrete, heterogeneous domains of phospholipids, sphingolipids, and sterols that are present in the cell membrane. They are responsible for conducting cell signaling and maintaining lipid-protein functionality. Redox-stress-induced modifications to any of their components can severely alter the mechanics and dynamics of the membrane causing impairment to the lipid-protein functionality. Here, we report on the effect of sphingomyelin (SM) in controlling membrane permeability and its role as a regulatory lipid in the presence of nitric oxide (NO). Force spectroscopy and atomic force microscopy imaging of raft-like phases (referring here to the coexistence of “liquid-ordered” and “liquid-disordered” phases in model bilayer membranes) prepared from lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC):SM:cholesterol (CH) (at three ratios) showed that the adhesion forces to pull the tip out of the membrane increased with increasing SM concentration, indicating decreased membrane permeability. However, in the presence of NO radical (1 and 5 μM), the adhesion forces decreased depending on SM concentration. The membrane was found to be stable at the ratio POPC:SM:CH (2:1:1) even when exposed to 1 μM NO. We believe that this is a critical ratio needed by the raft-like phases to maintain homeostasis under stress conditions. The stability could be due to an interplay existing between SM and CH. However, at 5 μM NO, membrane deteriorations were detected. For POPC:SM:CH (2:2:1) ratio, NO displayed a pro-oxidant behavior and damaged the membrane at both radical concentrations. These changes were reflected by the differences in the height profiles of the raft-like phases observed by atomic force microscopy imaging. Malondialdehyde (a peroxidation product) detection suggests that lipids may have undergone lipid nitroxidation. The changes were instantaneous and independent of radical concentration and incubation time. Our study underlines the need for identifying appropriate ratios in the lipid rafts of the cell membranes to withstand redox imbalances caused by radicals such as NO.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Impact of the Rat R5G, Y10F, and H13R Mutations on Tetrameric Aβ42 β-Barrel in a Lipid Bilayer Membrane Model

Three amino acid substitutions distinguish rat and human Aβ42 peptides and contribute to the difference in toxicity properties. Indeed, aged rodents rarely develop the characteristic lesions of Alzheimer's disease in contrast to humans. Both peptides form, however, amyloid fibrils in buffer solution, but their affinities to the membrane vary. In particular, there is experimental evidence that the rat Aβ42 peptide does not induce Ca2+ fluxes in cells. We recently designed a tetrameric β-barrel structure and showed that this model is severely destabilized for Aβ40 human compared to its Aβ42 human counterpart, explaining the absence of ionic currents of Aβ40 in planar lipid bilayers. In this study, we asked whether our model is destabilized for the rat Aβ42 peptide by using extensive replica exchange molecular dynamics simulation in a dipalmitoylphosphatidylcholine (DPPC) lipid bilayer membrane. Our results show that the much lower propensity of aged rodents to develop Alzheimer's disease symptoms might be correlated to its tetrameric β-barrel stability in the cell membrane.

A Comparative Study on DMSO-Induced Modulation of the Structural and Dynamical Properties of Model Bilayer Membranes

Modulating the structures and properties of biomembranes via permeation of small amphiphilic molecules is immensely important, having diverse applications in cell biology, biotechnology, and pharmaceuticals, because their physiochemical and biological interactions lead to new pathways for transdermal drug delivery and administration. In this work, we have elucidated the role of dimethyl sulfoxide (DMSO), broadly used as a penetration-enhancing agent and cryoprotective agent on model lipid membranes, using a combination of fluorescence microscopy and time-resolved fluorescence spectroscopy. Spatially resolved fluorescence lifetime imaging microscopy (FLIM) has been employed to unravel how the fluidity of the DMSO-induced bilayer regulates the structural alteration of the vesicles. Moreover, we have also shown that the dehydration effect of DMSO leads to weakening of the hydrogen bond between lipid headgroups and water molecules and results in faster solvation dynamics as demonstrated by femtosecond time-resolved fluorescence spectroscopy. It has been gleaned that the water dynamics becomes faster because bilayer rigidity decreases in the presence of DMSO, which is also supported by time-resolved rotational anisotropy measurements. The enhanced diffusivity and increased membrane fluidity in the presence of DMSO are further ratified at the single-molecule level through fluorescence correlation spectroscopy (FCS) measurements. Our results indicate that while the presence of DMSO significantly affects the 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphatidylcholine (DPPC) bilayers, it has a weak effect on 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) vesicles, which might explain the preferential interaction of DMSO with the positively charged choline group present in DMPC and DPPC vesicles. The experimental findings have also been further verified with molecular dynamics simulation studies. Moreover, it has been observed that DMSO is likely to have a differential effect on heterogeneous bilayer membranes depending on the structure and composition of their headgroups. Our results illuminate the importance of probing the lipid structure and composition of cellular membranes in determining the effects of cryoprotective agents.

Molecular dynamics simulations and experimental studies reveal differential permeability of withaferin-A and withanone across the model cell membrane

Poor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs—withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that whereas withaferin-A, could proficiently transverse through the model membrane, withanone showed weak permeability. The free energy profiles for the interaction of withanolides with the model bilayer membrane revealed that whereas the polar head group of the membrane caused high resistance for the passage of withanone, the interior of the membrane behaves similarly for both withanolides. The solvation analysis further revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force for its high permeability; it interacted with the phosphate group of the membrane that led to its smooth passage across the bilayer. The computational predictions were tested by raising and recruiting unique antibodies that react to withaferin-A and withanone. The time-lapsed analyses of control and treated cells demonstrated higher permeation of withaferin-A as compared to withanone. The concurrence between the computation and experimental results thus re-emphasised the use of computational methods for predicting permeability and hence bioavailability of natural drug compounds in the drug development process.

The Effect of Cholesterol on the Voltage-Current Characteristics of Tethered Lipid Membranes.

In this study, a lipid bilayer membrane model was used, in which the bilayer is tethered to a solid substrate with molecular tethers. The V-I characteristics of the lipid bilayers were found to be non-linear which suggests the presence of pores that are voltage-dependent. At high applied voltages, the conductance reached a limiting value, presumably indicating a limit on the maximum pore size. A decrease in the spacing between tethers (increasing tether density) caused a decrease in the membrane's conductance at high applied voltage, which is consistent with the maximum pore size being determined by the spacing between the tethers. The inclusion of 10M% cholesterol within the membrane lipid caused a decrease in the membrane conductance. However, the inclusion of higher levels of cholesterol increased the membrane conductance.