Flip-flop Rate Research Articles

Studying lipid flip-flop in asymmetric liposomes using 1H NMR and TR-SANS.

The specific spatial and temporal distribution of lipids in membranes play a crucial role in determining the biochemical and biophysical properties of the system. In nature, the asymmetric distribution of lipids is a dynamic process with ATP-dependent lipid transporters maintaining asymmetry, and passive transbilayer diffusion, that is, flip-flop, counteracting it. In this chapter, two probe-free techniques, 1H NMR and time-resolved small angle neutron scattering, are described in detail as methods of investigating lipid flip-flop rates in synthetic liposomes that have been generated with an asymmetric bilayer composition.

Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip-Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine.

Although lateral and inter-leaflet lipid-lipid interactions in cell membranes play roles in maintaining asymmetric lipid bilayers, the molecular basis of these interactions is largely unknown. Here, we established a method to determine the distribution ratio of phospholipids between the outer and inner leaflets of asymmetric large unilamellar vesicles (aLUVs). The trimethylammonium group, (CH3)3N+, in the choline headgroup of N-palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state 1H NMR (MAS-ss-1H NMR). PSM and DOPC have the same headgroup structure, but one phospholipid was selectively observed by deuterating the trimethylammonium group of the other phospholipid. The addition of Pr3+ to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH3)3N+ group in the outer leaflet from that in the inner leaflet, which allowed estimation of the inter-leaflet distribution ratio of the unlabeled lipid in aLUVs. Using this method, we evaluated the translocation of PSM and DOPC between the outer and inner leaflets of the cholesterol-containing aLUVs, with PSM and DOPC mostly distributed in the outer and inner leaflets, respectively, immediately after aLUV preparation; their flip and flop rates were approximately 2.7 and 6.4 × 10-6 s-1, respectively. During the passive symmetrization of aLUVs, the lipid translocation rate was decreased due to changes in the membrane order, probably through the formation of the registered liquid-ordered domains. Comparison of the result with that of symmetric LUVs revealed that lipid asymmetry may not significantly affect the lipid translocation rates, while the lateral lipid-lipid interaction may be a dominant factor in lipid translocation under these conditions. These findings highlight the importance of considering the effects of lateral lipid interactions within the same leaflet on lipid flip-flop rates when evaluating the asymmetry of phospholipids in the cell membrane.

Renovating a double fence with or without notifying the next door and across the street neighbors: why the biogenic cytoplasmic membrane of Gram-negative bacteria display asymmetry?

The complex two-membrane organization of the envelope of Gram-negative bacteria imposes an unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on the cytoplasm facing leaflet of the cytoplasmic (inner) membrane (IM), across the IM and between the IM and outer membrane (OM). Balanced growth of two membranes and continuous loss of phospholipids in the periplasmic leaflet of the IM as metabolic precursors for envelope components and for translocation to the OM requires a constant supply of phospholipids in the IM cytosolic leaflet. At present we have no explanation as to why the biogenic E. coli IM displays asymmetry. Lipid asymmetry is largely related to highly entropically disfavored, unequal headgroup and acyl group asymmetries which are usually actively maintained by active mechanisms. However, these mechanisms are largely unknown for bacteria. Alternatively, lipid asymmetry in biogenic IM could be metabolically controlled in order to maintain uniform bilayer growth and asymmetric transmembrane arrangement by balancing temporally the net rates of synthesis and flip-flop, inter IM and OM bidirectional flows and bilayer chemical and physical properties as spontaneous response. Does such flippase-less or 'lipid only", 'passive' mechanism of generation and maintenance of lipid asymmetry exists in the IM? The driving force for IM asymmetry can arise from the packing requirements imposed upon the bilayer system during cell division through disproportional distribution of two negatively curved phospholipids, phosphatidylethanolamine and cardiolipin, with consistent reciprocal tendency to increase and decrease lipid order in each membrane leaflet respectively.

Distinct roles of graphene and graphene oxide nanosheets in regulating phospholipid flip-flop

Two-dimensional (2D) nanomaterials, such as graphene nanosheets (GNs) and graphene oxide nanosheets (GOs), could adhere onto or insert into a biological membrane, leading to a change in membrane properties and biological activities. Consequently, GN and GO become potential candidates for mediating interleaflet phospholipid transfer. In this work, molecular dynamics (MD) simulations were employed to investigate the effects of GN and GO on lipid flip-flop behavior and the underlying molecular mechanisms. Of great interest is that GN and GO work in opposite directions. The inserted GN can induce the formation of an ordered nanodomain, which dramatically elevates the free energy barrier of flipping phospholipids from one leaflet to the other, thus leading to a decreased lipid flip-flop rate. In contrast, the embedded GO can catalyze the transport of phospholipids between membrane leaflets by facilitating the formation of water pores. These results suggest that GN may work as an inhibitor of the interleaflet lipid translocation, while GO may play the role of scramblases. These findings are expected to expand promising biomedical applications of 2D nanomaterials.

Investigating the cut-off effect of n-alcohols on lipid movement: a biophysical study.

Cellular membranes are responsible for absorbing the effects of external perturbants for the cell's survival. Such perturbants include small ubiquitous molecules like n-alcohols which were observed to exhibit anesthetic capabilities, with this effect tapering off at a cut-off alcohol chain length. To explain this cut-off effect and complement prior biochemical studies, we investigated a series of n-alcohols (with carbon lengths 2-18) and their impact on several bilayer properties, including lipid flip-flop, intervesicular exchange, diffusion, membrane bending rigidity and more. To this end, we employed an array of biophysical techniques such as time-resolved small angle neutron scattering (TR-SANS), small angle X-ray scattering (SAXS), all atomistic and coarse-grained molecular dynamics (MD) simulations, and calcein leakage assays. At an alcohol concentration of 30 mol% of the overall lipid content, TR-SANS showed 1-hexanol (C6OH) increased transverse lipid diffusion, i.e. flip-flop. As alcohol chain length increased from C6 to C10 and longer, lipid flip-flop slowed by factors of 5.6 to 32.2. Intervesicular lipid exchange contrasted these results with only a slight cut-off at alcohol concentrations of 30 mol% but not 10 mol%. SAXS, MD simulations, and leakage assays revealed changes to key bilayer properties, such as bilayer thickness and fluidity, that correlate well with the effects on lipid flip-flop rates. Finally, we tie our results to a defect-mediated pathway for alcohol-induced lipid flip-flop.

Microscopic model of spin flip-flop processes in crystals doped by rare-earth ions

Flip-flop processes due to magnetic dipole-dipole interaction between neighboring ions in crystals doped by rare-earth ions are one of the mechanisms of relaxation between hyperfine levels. Modeling of this mechanism has so far been macroscopic, characterized by an average rate describing the relaxation of all ions. Here, however, we present a microscopic model of flip-flop interactions between individual nuclear spins of dopant ions. Every ion is situated in a unique local environment in the crystal, where each ion has different distances and a unique orientation relative to its nearest neighbors, as determined by the lattice structure. Thus each ion has a unique flip-flop rate and the collective relaxation dynamics of all ions in a bulk crystal is a sum of many exponential decays, giving rise to a distribution of rates rather than a single average decay rate. We employ this model to calculate flip-flop rates in ${\mathrm{Pr}}^{3+}$:${\mathrm{Y}}_{2}{\mathrm{SiO}}_{5}$ and show experimental measurements of population decay of the ground state hyperfine levels at $\ensuremath{\sim}2$ K. We also present a method to measure rates of individual transitions from hole burning spectra that requires significantly fewer fitting parameters in theoretical rate equations compared to earlier work. Furthermore, we measure the effect of external magnetic field on the flip-flop rates and observe that the rates slow down by two orders of magnitude in a field of 5--10 mT.

Multilamellar Liposomes as a Model for Biological Membranes: Saturation Recovery EPR Spin-Labeling Studies.

EPR spin labeling has been used extensively to study lipids in model membranes to understand their structures and dynamics in biological membranes. The lipid multilamellar liposomes, which are the most commonly used biological membrane model, were prepared using film deposition methods and investigated with the continuous wave EPR technique (T2-sensitive spin-labeling methods). These investigations provided knowledge about the orientation of lipids, their rotational and lateral diffusion, and their rate of flip-flop between bilayer leaflets, as well as profiles of membrane hydrophobicity, and are reviewed in many papers and book chapters. In the early 1980s, the saturation recovery EPR technique was introduced to membrane studies. Numerous T1-sensitive spin-label methods were developed to obtain detailed information about the three-dimensional dynamic membrane structure. T1-sensitive methods are advantageous over T2-sensitive methods because the T1 of spin labels (1–10 μs) is 10 to 1000 times longer than the T2, which allows for studies of membrane dynamics in a longer time–space scale. These investigations used multilamellar liposomes also prepared using the rapid solvent exchange method. Here, we review works in which saturation recovery EPR spin-labeling methods were applied to investigate the properties of multilamellar lipid liposomes, and we discuss their relationships to the properties of lipids in biological membranes.

Influence of very-long-chain polyunsaturated fatty acids on membrane structure and dynamics

The unique attributes of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), their long carbon chains (n > 24) and high degree of unsaturation, impart unique chemical and physical properties to this class of fatty acids. The changes imparted by VLC-PUFA 32:6 n-3 on lipid packing and the compression moduli of model membranes were evaluated from π-A isotherms of VLC-PUFA in 1,2-distearoyl-sn-3-glycero-phosphocholine (DSPC) lipid monolayers. To compare the attractive or repulsive forces between VLC-PUFA and DSPC lipid monolayers, the measured mean molecular areas (MMAs) were compared with the calculated MMAs of an ideal mixture of VLC-PUFA and DSPC. The presence of 0.1, 1, and 10 mol % VLC-PUFA shifted the π-A isotherm to higher MMAs of the lipids comprising the membrane and the observed positive deviations from ideal behavior of the mixed VLC-PUFA:DSPC monolayers correspond to repulsive forces between VLC-PUFAs and DSPC. The MMA of the VLC-PUFA component was estimated using the measured MMAs of DSPC of 47.1 ± 0.7 Å2/molecule, to be 15,000, 1100, and 91 Å2/molecule at 0.1, 1, and 10 mol % VLC-PUFA:DSPC mixtures, respectively. The large MMAs of VLC-PUFA suggest that the docosahexaenoic acid tail reinserts into the membrane and adopts a nonlinear structure in the membrane, which is most pronounced at 0.1 mol % VLC-PUFA. The presence of 0.1 mol % VLC-PUFA:DSPC also significantly increased the compression modulus of the membrane by 28 mN/m compared with a pure DSPC membrane. The influence of VLC-PUFA on lipid “flip-flop” was investigated by sum-frequency vibrational spectroscopy. The incorporation of 0.1 mol % VLC-PUFA increased the DSPC flip-flop rate fourfold. The fact that VLC-PUFA promotes lipid translocation is noteworthy as retinal membranes require a high influx of retinoids which may be facilitated by lipid flip-flop.

Using cyclodextrins to produce asymmetric liposomes, quantify lipid-lipid interactions and rates of transbilayer lipid flip-flop

Due to their bilayer structure, liposomes are prevalent model systems for studies of biophysical processes on plasma membranes, which naturally show lipid asymmetry. Phosphatidylethanolamine (PE), for example, occurs mainly in the inner leaflet and phosphatidylglycerol (PG) in the outer leaflet of prokaryotic membranes. Cyclodextrins (cyd) are widely used to extract, insert, or exchange lipids from or into the outer leaflet of liposomes or cell membranes. This strategy is, for example, playing a key role in several protocols to produce lipid-asymmetric liposomes.

Role of cholesterol flip-flop in oxidized lipid bilayers

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0–50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol’s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.

On the Structure and Flip-flop of Free Docosahexaenoic Acid in a Model Human Brain Membrane.

Among the omega-3 fatty acids, docosahexaenoic acid (DHA, sn22:6) is particularly vital in human brain cell membranes. There is considerable interest in DHA because low-level DHA has been associated with declined cognitive function and poor visual acuity. In this work, atomistic molecular dynamics simulations were used to investigate the effects of free protonated DHA (DHAP) in molar fractions of 0, 17, 30, and 38% in a realistic model of a healthy brain cell membrane comprising 26 lipid types. Numerous flip-flop events of DHAP were observed and categorized as successful or aborted. Novel use of the machine learning technique, density-based spatial clustering of applications with noise (DBSCAN), effectively identified flip-flop events by way of clustering. Our data show that increasing amounts of DHAP in the membrane disorder the bilayer packing, fluidize the membrane, and increase the rates of successful flip-flop from k = 0.2 μs-1 (17% DHAP) to 0.8 μs-1 (30% DHAP) and to 1.3 μs-1 (38% DHAP). In addition, we also provided a detailed understanding of the flip-flop mechanism of DHAP across this complex membrane. Interestingly, we noted the role of hydrogen bonds in two distinct coordinated flip-flop phenomena between two DHAP molecules: double flip-flop and assisted flip-flop. Understanding the effects of various concentrations of DHAP on the dynamics within a lipid membrane and the resulting structural properties of the membrane are important when considering the use of DHAP as a dietary supplement or as a potential therapeutic.

Molecular dynamics simulation study of the positioning and dynamics of α-tocopherol in phospholipid bilayers.

Using molecular dynamics simulations, we investigate the interaction of α-tocopherol (α-toc) with dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (POPE) lipid bilayers. The goal is to develop a better understanding of the positioning and orientation of α-toc inside the bilayers; properties of significant relevance to α-toc anti-oxidant activity. We investigated bilayer systems with 128 lipids in the presence of either single or 14 α-toc molecules. The single α-toc bilayer systems were investigated via biased MD simulations in which the potential of mean force (PMF) and diffusivity were obtained as functions of the distance between α-toc head group and bilayer center. The higher α-toc concentration systems were investigated with unbiased MD simulations. For all four bilayers at both concentrations, the simulations show that the most probable location of the α-toc hydroxyl group is just below the lipid carbonyl group. Overall, the simulation results are in good agreement with existing experimental data except for the DMPC bilayer system for which some experiments predict α-toc to be located closer to bilayer center. The flip-flop frequency calculated shows that the α-toc flip-flop rate is sensitive to bilayer lipid type. In particular, α-toc has a much lower flip-flop rate in a POPE bilayer compared to the three PC lipid bilayers due to the smaller area per lipid in the POPE bilayer. For DMPC and POPC, the α-toc flip-flop rates are significantly higher at higher α-toc concentration and this appears to be related to the local structural disruption caused by α-toc clusters spanning the bilayer.

The effects of the lipid type on the spatial arrangement and dynamics of cholesterol in binary component lipid membranes.

Intermolecular interactions between cholesterol and lipids in cell membranes, which play critical roles in cellular processes such as the formation of nano-domains, depend on the molecular structure of the lipids. The diffusion and the spatial arrangement of cholesterol within the lipid membranes also change with the type of lipids. For example, the flip-flop, an important transport mechanism for cholesterol in the membranes, can be facilitated significantly by the presence of unsaturated lipids. However, how the structure of lipids affects the spatial arrangement and the dynamics of cholesterol remains elusive at a molecular level. In this study, we investigate the effects of lipid-cholesterol interactions on the spatial arrangement and the dynamics of cholesterol. We perform molecular dynamics simulations for the binary component membranes of lipids and cholesterol. We employ seven different kinds of lipids by changing either the degree of a saturation level or the length of lipid tails. We find from our simulations that the rate of cholesterol flip-flop is enhanced as the lipids are either less saturated or shorter, which is consistent with previous studies. Interestingly, when the lipid tails are fully saturated and sufficiently long, the center in between two leaflets becomes metastable for cholesterol to stay at. Because the cholesterol at the membrane center diffuses faster than that within leaflets, regardless of the lipid type, such an emergence of the metastable state (in terms of the cholesterol position) complicates the cholesterol diffusion significantly.

Asymmetric desorption of lipid oxidation products induces membrane bending.

Lipid oxidation, detected in metabolic processes, is induced in excess when the cellular membrane suffers extra oxidative stress. Lipid oxidation can compromise biomembrane function in part through perturbations of lipid packing, membrane permeability, and morphology. Two major types of oxidation products, one with a partially truncated lipid tail with a hydrophilic group at the tail-end, and secondly, a lysolipid (with one of the chains completely truncated) can disturb the membrane bilayer packing significantly. However, they also have an increased tendency to desorb from the membrane. In this study we investigated desorption kinetics of two characteristic lipid oxidation products (PAzePC and 18 : 1 LysoPC) from a model membrane system, and we evaluated the consequences of this process on membrane shape transitions. Using a microfluidic chamber coupled with micropipette aspiration, we observed the incorporation of the two lipids into the membrane of a giant unilamellar vesicle (GUV) and further determined their desorption rates, association rates and flip-flop rates. For both lipids, the desorption is on the time scale of seconds, one to two orders of magnitude faster than their flipping rates. Dilution of the outer solution of the GUVs allowed asymmetric desorption of these two lipids from the GUVs. This process induced lipid number asymmetry and charge asymmetry, specifically for PAzePC containing GUVs, and caused membrane tubulation. Our results indicate that the desorption of lipid oxidation products can alter the local structure of biomembranes and result in morphological changes that may relate to membrane function.

Vesicle deformation and division induced by flip-flops of lipid molecules.

We investigated the deformation of small unilamellar vesicles (SUVs) induced by flip-flops of lipids using coarse-grained molecular dynamics simulations. In the case of single-component SUVs composed of zero spontaneous curvature lipids (ZLs), the flip-flop of ZLs deformed stomatocyte-shaped SUVs into an oblate shape, whereas pear-shaped SUVs were deformed into a prolate shape. These two equilibrium shapes comply with the local minima of elastic energy. In the case of binary vesicles composed of ZLs and negative spontaneous curvature lipids (NLs), the vesicle deformation pathway depended on the initial NL distribution in the bilayer. If the initial difference in the NL concentration between the outer and inner leaflets was small, the flip-flop of ZLs and NLs rapidly deformed pear-shaped SUVs into an equilibrium prolate shape. On the other hand, when NLs were localised in the inner leaflet, the flip-flop of ZLs and NLs deformed pear-shaped SUVs into a limiting shape and then induced vesicle division. Because the flip-flop rate of NLs is much faster than that of ZLs, the total free energy was first relaxed by the flip-flop of NLs and then by that of ZLs. This kinetic effect is responsible for the observed vesicle division induced by flip-flops.

Membrane Perturbation and Lipid Flip-Flop Mediated by Graphene Nanosheet.

Graphene nanosheets (GNs) may spontaneously insert into cell membranes and perturb the dynamic organization of the surrounding lipid bilayer. Understanding the interaction between GNs and cell membranes is vital for learning how to avoid adverse effects and nanomedical applications. To better understand the nature of such perturbations, we performed molecular dynamics simulations to provide molecular details about the molecular mechanism. In this study, we observed two typical interaction states of the GN-membrane systems. Both states have different effects on the cell membrane (lipid density, membrane thickness, and the mobility of phospholipids). Of great interest is that the insertion of GNs could generate a liquid-ordered domain and dramatically reduce the rate of lipid flip-flop. A similar phenomenon could be found in the GN adhesion states. Thus, these results could facilitate molecular-level understanding of the cytotoxicity of nanomaterials and help future studies on designing personalized drugs and therapeutics for diseases.

Effect of alcohol on the phase separation in model membranes

The discovery of coexisting liquid-ordered and liquid-disordered phases in multicomponent lipid bilayers has received widespread attention due to its potential relevance for biological systems. One of the many open questions is how the presence of additional components affects the nature of the coexisting phases. Of particular interest is the addition of alcohols because their anesthetic properties may arise from modulating bilayer behavior. We use coarse-grained Molecular Dynamics simulations to gain insight into the partitioning preferences of linear n-alcohols into ordered and disordered bilayers alongside their effects on local membrane structure. We find that alcohols cause only small changes to membrane composition alongside a lack of significant effects on membrane thickness and lipid tail order. Cholesterol and n-alcohol trans-bilayer motion is measured and found to be near or within the range of previous atomistic results. The cholesterol flip-flop rates increase with both n-alcohol length and concentration for octanol, dodecanol, and hexadecanol, indicating a decrease in lipid order. Umbrella sampling simulations of removing cholesterol from tertiary membranes find no significant difference with or without n-alcohols at various concentrations. Simulations of a phase-separated bilayer show that octanol preferentially partitions into the liquid-disordered phase in a ratio of approximately 3:1 over the liquid-ordered phase. Furthermore, partition coefficients of alcohol in single-phase membranes show a preference for longer alcohols (dodecanol and hexadecanol) to partition preferentially into the liquid-ordered phase, while decreasing the length of the alcohol reverses this trend. Our work tests experimental results while also investigating the ability for coarse-grained MARTINI simulations to capture minute differences in model membrane spatial arrangements on the nanoscale level.

Composition dependence of cholesterol flip-flop rates in physiological mixtures

Unbiased, all-atom simulations of mixtures representative of the inner and outer leaflets of a mammalian red blood cell and a synaptic vesicle reveal many cholesterol flip-flop events over the 5 μsec duration of the simulations. Enough events are observed for a direct estimate of the flip-flop rate. Slower rates are found in more ordered membranes, and faster rates in more disordered membranes, consistent with earlier reports in the literature. However, the rates found here are neither as fast as the fastest nor as slow as the slowest rates obtained by previous simulations. The difference likely stems from the compositions studied here, which unlike previous work include exclusively lipids with differing acyl chains, as observed in mammalian lipidomes.

Simulations of Asymmetric Membranes Illustrate Cooperative Leaflet Coupling and Lipid Adaptability

Biological membranes are composed of lipid bilayers that are often asymmetric with regards to the lipid composition and/or aqueous solvent they separate. Studying lipid asymmetry both experimentally and computationally is challenging. Molecular dynamics simulations of lipid bilayers with asymmetry are difficult due to finite system sizes and time scales accessible to simulations. Due to the very slow flip-flop rate for phospholipids, one must first choose how many lipids are on each side of the bilayer, but the resulting bilayer may be unstable (or metastable) due to differing tensile and compressive forces between leaflets. Here we use molecular dynamics simulations to investigate a number of different asymmetric membrane systems, both with atomistic and coarse-grained models. Asymmetries studied include differences in number of lipids, lipid composition (unsaturated and saturated tails and different headgroups), and chemical gradients between the aqueous phases. Extensive analysis of the bilayers’ properties such as area per lipid, density, and lateral pressure profiles are used to characterize bilayer asymmetry. We also address how cholesterol (which flip-flops relatively quickly) influences membrane asymmetries. Our results show how each leaflet is influenced by the other and can mitigate the structural changes to the bilayer overall structure. Cholesterol can respond to changes in bilayer asymmetry to alleviate some of the effect on the bilayer structure, but that will alter its leaflet distribution, which in turn affects its chemical potential. Ionic imbalances are shown to have a modest change in bilayer structure, despite large changes in the electrostatic potential. Bilayer asymmetry can also induce a modest electrostatic potential across the membrane. Our results highlight the importance of membrane asymmetry on bilayer properties, the influence of lipid headgroups, tails and cholesterol on asymmetry, and the ability of lipids to adapt to different environments.

Electrochemical Measurement of Cholesterol Flip-Flop in Plasma Membrane at Single Cells.

Here, a microelectrode approach is established to measure the flip-flop rate of cholesterol in plasma membranes at single living cells. The initial validation is performed in a modeled phospholipid bilayer positioned at an interconnecting hole between two compartments, in which cholesterol in one compartment diffuses into the other one through a flip-flop movement in the bilayer and is then detected by a cholesterol oxidase-modified microelectrode. As compared with the time (140 ± 28 s) for free cholesterol transport in absence of the bilayer, a prolonged time (702 ± 42 s) is needed to observe the current increase in the presence of the bilayer. The difference in the time (562 s) gives the estimated flip-flop time of cholesterol in the bilayer. The position of the microelectrode in contact with a living cell and the injection of cholesterol inside the cell are further applied to measure the cholesterol flip-flop in the plasma membrane. The average time (1183 ± 146 s) is obtained to observe an additional current increase at the microelectrode, which reflects the cholesterol flip-flop rate in plasma membranes in single living cells. All these results support the establishment of this microelectrode approach for the study of the cholesterol flip-flop process in lipid membranes.