Water Permeability across Symmetric and Asymmetric Droplet

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Water Permeability across Symmetric and Asymmetric Droplet Interface Bilayers: Interaction of Cholesterol Sulfate with DPhPC Peter J. Milianta, Michelle Muzzio, Jacqueline Denver, Geoffrey Cawley, and Sunghee Lee* Department of Chemistry, Iona College, 715 North Avenue, New Rochelle, New York 10801, United States S Supporting Information *

ABSTRACT: Cellular membranes employ a variety of strategies for controlling the flow of small molecules into the cytoplasmic space, including incorporation of sterols for modulation of permeability and maintenance of lipid asymmetry to provide both sides of the membrane with differing biophysical properties. The specific case of cholesterol asymmetry, especially, is known to have profound effects in neurological cellular systems. Synthetic membrane models that can readily determine valuable physical parameters, such as water transport rates, for sterolcontaining membranes of defined lipid composition remain in demand. We report the use of the droplet interface bilayer (DIB), composed of adherent aqueous droplets surrounded by a lipid monolayer and immersed in a hydrophobic medium, for measurement of water permeability across the membrane, with rapid visualization and ease of experimental setup. We studied droplet bilayer membranes composed of the prototypical synthetic membrane lipid (i.e., the archaeal lipid DPhPC) as well as of symmetric and asymmetric DIBs formed by DPhPC and sodium cholesterol sulfate (S-Chol). The presence of S-Chol in DPhPC in symmetric DIB reduced the passive water permeability rate (Pf) at all concentrations and increased the activation energy (Ea) to 17−18 kcal/mol. When only one side of the DIB contains S-Chol (asymmetric DIB), an Ea of 14−15 kcal/mol was obtained, a value intermediate that of pure lipid and symmetrical DIB containing lipid and S-Chol. Our data are consistent with a capability for regulation of water transport by one leaflet independent of the other. The engineering of our various systems is believed to have implications for garnering detailed knowledge regarding the transport of small moieties across bilayers in a wide variety of lipid systems.

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explored, including effects on water permeability.8 Additionally, asymmetric transmembrane distribution of membrane sterol has recently been an extensive topic of study.9−11 The various forms of lipid asymmetry have consequences for membrane structure and function. To improve understanding of how these consequences arise, experimental and computational studies have investigated asymmetry from the standpoint of interleaflet coupling, finding very weak coupling12 in some cases to various types of coupling within a bilayer,13 leading to change in its structural and physical properties. Most previous studies of water permeability have employed systems such as liposomes14 and various supported lipid membranes.15 Recently, however, we have demonstrated a method using a droplet interface bilayer (DIB) as a membrane model for determination of water permeability.16 The DIB method is a simple technique for constructing a stable lipid bilayer at the interface of two water droplets immersed in oil where bilayer-forming lipids can be introduced either in water droplet (lipid-in) or oil solvent (lipid-out).17,18 The DIB-based water permeability measurement technique allows rapid

INTRODUCTION Cells in living systems are surrounded by a membrane which functions as a barrier that is semipermeable to the cellular surroundings, so as to maintain control of cell contents. The ability to understand membrane permeation is of critical importance, as the transport of small molecules across bilayer membranes has significant implications for cellular physiology and homeostasis.1,2 Mechanisms for molecular movement across membranes can be categorized as either constituting active transport, where molecules move across the membrane in the direction opposed to their concentration gradient upon an input of energy (usually assisted by transmembrane proteins), or passive transport, which is entropically driven. The latter mode is important for a wide range of neutral species. Among the several types of molecules transported by passive permeation, the permeability of water molecules across cellular membranes has been widely investigated, in order to understand how the rate of water flow is controlled as a function of lipid bilayer composition, structure, and property in various cellular membranes.3,4 In biological systems, cell membranes contain different lipid compositions in the inner and outer leaflets of the bilayer.5−7 While the asymmetric lipid bilayer is dominant for a variety of cell types, the roles of bilayer asymmetry on various membrane properties are still being © XXXX American Chemical Society

Received: July 25, 2015 Revised: October 19, 2015

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Langmuir visualization and determination of the water transport rate across biological membranes.16 To achieve a better understanding of the interaction of lipid acyl tails with sterol on water permeability, and to examine any differences in water transport for putative symmetric and asymmetric membranes, we use the DIB method to investigate a membrane of a phospholipid (diphytanoylphosphocholine (DPhPC)) with and without added cholesterol sulfate (S-Chol) and compare water permeabilities. DPhPC belongs to the family of archaeal lipids and has been extensively used as the lipid of choice in synthetic bilayer formation.19 While DPhPC is not ordinarily found in human or animal membranes,20 its highly branched and saturated hydrocarbon tail structure affords increased resistance to oxidation.21,22 Also, DPhPC exists in the fluid phase over a wide range of temperatures, and it is this fluid phase which has relevance to mammalian biological systems.22,23 Additionally, DPhPC bilayers generally do not permit ions to leak in the absence of a pore/ion channel,22,24 which can be useful for studies on channel proteins. Finally, DPhPC has a very high solubility in the hydrocarbon solvents commonly used for DIBs (e.g., hexadecane, squalane), making it convenient for experimental use. Sterol molecules play an important role in biological membranes, both plant- and animal-based.25,26 S-Chol is a naturally occurring sterol, namely, a sulfated derivative of cholesterol, which is found as a membrane component that is widely distributed in a variety of mammalian tissues. It has been known to be abundant in spermatozoa membranes, erythrocyte membranes, and the uppermost layer of human epidermis (the “stratum corneum”). Previous reports on the physiological role of S-Chol27 and experimental28−30 and theoretical31 studies suggest that while S-Chol generally represents a small percentage of total membrane sterols, it is known to contribute to membrane stability and interact with phospholipids in a manner similar to that observed for cholesterol. In this study, we report water permeability across symmetric and asymmetric DPhPC membranes created by the DIB method and the effect of added S-Chol in varying concentration. We use the “lipid-in” method for DIB formation to create membranes from DPhPC and a mixture of DPhPC with S-Chol for symmetric and asymmetric DIB. Earlier studies by Bayley have demonstrated an asymmetric DIB by introducing different vesicle compositions in a droplet pair.32 In our present work, membranes in which potential lipid asymmetry is induced were achieved by incorporating S-Chol in only one droplet of the droplet bilayer pair, but not the other droplet.

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Figure 1. Structure of DPhPC and S-Chol molecules. 4−5 mM in aqueous dispersion. Aqueous phases were either pure water or osmolyte solutions. Rehydrated and resuspended lipid solution is extruded with 21 passes through a membrane filter (Avanti mini-extruder with 0.1 μm polycarbonate membrane filter). Aqueous solutions using osmolytes (NaCl at nominally 0.1 M) were prepared from a high-grade of commercial salts dissolved in purified, deionized water (18.2 MΩ·cm), obtained using a Millipore water purification system (Direct Q-3). The osmolality (in mOsm/kg) of all solutions was measured by a vapor pressure osmometer. All solutions were prepared immediately prior to use. All experiments were carried out using a custom-built temperature-controlled microchamber which was thermostated via an external circulating water bath. This allows variation and control of temperature from 16 to 65 °C within ±1 °C accuracy. Hexadecane (n-C16H34) was used as immiscible oil solvent. The detailed description of the procedure for permeability measurements using DIB has been previously reported.16 In brief, a DIB is formed by contacting two aqueous droplets, each possessing a lipid monolayer, in an immiscible solvent. For the creation and manipulation of the droplets, we employed an inverted microscope (Nikon Eclipse Ti−S with halogen lamp) combined with two hydraulic micropipet manipulators (Narishige), supported on a vibration isolated workstation (Newport). As schematically shown in Figure 2, a DIB can be formed by having lipids present in aqueous

Figure 2. (a) DIB is formed with lipids present as vesicles within the aqueous droplet (lipid-in method); (b) when two osmotically imbalanced droplets were made to adhere at a bilayer, water transport immediately commenced and corresponding changes in droplet volume (dV/dt) over time is observed. (c) Micrograph of a DIB formed from DPhPC at 30 °C over the time course of ∼400 s. The scale bar on the image represents 50 μm.

MATERIALS AND METHODS

1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained from Avanti Polar Lipids (Alabaster, AL), and sodium cholesteryl sulfate (S-Chol) was obtained from Sigma. As shown in Figure 1, DPhPC possesses two identical saturated hydrocarbon chains with branches of four methyl groups (in positions 3, 7, 11, and 15). S-Chol is a negatively charged cholesterol analogue. The functionalization of the hydroxyl group in cholesterol with sulfate affords a charged and hydrated polar head group of large size.27,33 Droplet interface bilayers are formed by a method comprising a first step of preparing aqueous droplets containing lipids present as vesicles. In order to prepare a vesicle solution, chloroform solutions of pure DPhPC (or DPhPC mixed with S-Chol) are evaporated to make a dried film of lipid or mixed lipid, which is then rehydrated. Portions are adjusted to yield the final total lipid concentration of approximately

droplet (lipid-in) or alternatively in immiscible solvent (lipid-out) (but only the lipid-in method was used herein). When two osmotically imbalanced droplets, each covered with lipid monolayer, were made to adhere at a bilayer, water transport immediately commenced, and corresponding changes in droplet volume (dV/dt) over time is measured optically by microsopic observation; the behavior of the system follows the expression of eq 1: B

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Figure 3. (a) Osmotic water permeability coefficients (μm/s) of DIB membranes formed from DPhPC formed in C16 in the range of 30−40 °C. (b) Arrhenius plot of the natural log of the permeability coefficient (Pf) versus the reciprocal of absolute temperature of membranes formed from DPhPC.

dV (t ) = − Pf A(t )vw ΔC(t ) dt

values versus bilayer water permeability values obtained from other saturated but nonbranched phosphocholines of a similar length. For example, DMPC (dimyristoylphosphatidylcholine) vesicles have been reported to have a Pf value of ca. 80 μm/s at 30 °C,4 and DPPC (dipalmitoylphosphatidylcholine) vesicles have been reported a Pf of ca. 270 μm/s at 50 °C (at which DPPC is in a fluid phase).36 Thus, DPhPC seems to have a lower permeability than these comparable PC lipids. It is generally considered that water transport rates across bilayers correlate to the fluidity of the lipids from which the membrane is composed; in turn, this fluidity is largely dependent on the shape factor of the hydrocarbon tail group. There is enhanced permeability observed for lipids of relatively greater fluidity, while those of lower fluidity have lessened permeability, a phenomenon which may be attributable to tighter packing of lipids in assemblies of lipids having low fluidity. Many interpretations ascribe water permeation to the formation of porous voids in the tail groups due to random thermal fluctuations in chain packing.37 Recall that DPhPC is generally considered to be in a fluid state under the conditions we have studied (transition temperature, Tm < −120 °C). Therefore, as a first approximation, one might consider the permeation behavior of DPhPC to be dominated by its phase behavior.38 However, the contribution of the branching of the tail groups may also be significant. DPhPC possess two identical saturated hydrocarbon chains having branches of four methyl groups (in positions 3, 7, 11, and 15) and a relatively large head group. Recent studies of DPhPC in a Langmuir monolayer show a relatively high value for the molecular area of 78.3 ± 5.1 Å2/molecule vs 47.4 Å2/molecule for DPPC.39 DPhPC molecules are slightly tapered, but once they are introduced in a membrane they tend to adopt a cylindrical shape by the formation of interdigitated structures by the methyl-functionalized chains.40,41 Possibly owing to this interdigitation, DPhPC forms highly packed and ordered membranes, reflected by their low surface tension (32−37 mN/m vs 54−56 mN/m for many other phospholipids at the air−water interface).42 Using molecular dynamics (MD) simulations, it has been demonstrated that DPhPC molecules have slower rotational and translational motions compared to other linear chain PC molecules. It has been suggested that the high structural stability and low permeability of the branched DPhPC bilayer would be closely related to this slow conformational motion of the branched hydrophobic chain due to the chain entrapment between the lateral neighboring lipid molecules.41 Further analysis of the cavity distribution of DPhPC bilayers

(1)

where A is the geometric bilayer area, νw is the molar volume of water (18 mL/mol), ΔC(t) is the osmolality gradient between two droplets, and Pf is the bilayer permeability coefficient of water. The DIB can be employed as a convenient model membrane to rapidly explore subtle structural effects on bilayer water permeability.16 All data presented in this paper are an average (n ≥ 10) of individual permeability runs, each of which took place over a time course (∼5−10 min) for osmotic water movement across the droplet bilayer, during which time the droplet contact area remains constant. The detailed methodology for how to extract permeability coefficient is shown in the Supporting Information. All experiments are video recorded using a camera (Andor Zyla sCMOS) directly attached to the microscope. The recorded videos and images were postanalyzed to measure the dimension of droplets and contact area using custom-built image analysis software. All droplet pairs had substantially the same initial size relative to each other, in the diameter range of 100 ± 5 μm. We chose our initial droplet size regime of ca. 50 μm radius based on several factors. First, our microscope objective configuration allows us to reliably measure osmotic size changes for droplets in this regime. All videos were collected with a pixel size of 0.16 μm using the entire field of 1920 × 1080 pixels. Thus, the uncertainty in the radius measurement is 0.32 μm (2 pixels × 0.16 μm/pixel). If the chosen droplet size is much smaller, then the uncertainty looms much larger. Conversely, if the droplet size is larger, then the time required to observe a given volume change scales as the initial volume (i.e., the time needed is proportional to r3, where r is initial radius).

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RESULTS AND DISCUSSION Temperature Dependence of Osmotic Water Permeability of DPhPC Membrane. Figure 3 shows osmotic water permeability coefficients Pf for the water transport process at varying temperatures in the range of 30−40 °C, for a DIB formed from DPhPC at 4−5 mM concentration in hexadecane (“C16”) . The permeability coefficients obtained at 30, 35, and 40 °C were 42 ± 3, 54 ± 4, and 80 ± 6 μm/s, respectively (Figure 3a). Also shown in Figure 3 is an Arrhenius plot from which the activation energy was determined (Figure 3b). In a plot of ln[Pf] versus 1/T (in K), the slope of the curve is equal to Ea/R, where Ea is the activation energy and R is the gas constant. The activation energies (Ea) for the water permeation process across DIB formed from DPhPC in C16 was determined to be 12.1 ± 0.8 kcal/mol. These observed values in permeability and activation energy are in comparable agreement with prior studies34,35 which gave Pf = 42.9 ± 3.0 μm/s at 25 °C, 70 ± 10 μm/s at 30 °C, and Ea = 12.1 kcal/mol for a DPhPC liposomes. It is worthwhile to compare these C

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aqueous solution which is saturated in hexadecane molecules, the work of Gruen implies both theoretically (via a statisticalmechanical mean-field model) and experimentally that a large proportion of the hexadecane molecules in the bilayer span the midplane, and the remainder are lined up parallel to phospholipid chains.47 More recently, Haines has obtained unambiguous evidence for the localization of squalene in lipid bilayers, employing neutron diffraction which clearly showed that the squalene lies predominantly in the bilayer center, parallel to the plane of the membrane, i.e., sandwiched between the two monolayers.48 For understanding what effect hydrocarbons at the midplace of the bilayer may have on water permeability, it may be instructive to take into account one of the leading theoretical models for permeability: the inhomogeneous solubility− diffusion mechanism (ISDM). This mechanism attempts to take into account both the thermodynamic (solubility) and kinetic (diffusion) aspects of the lipid bilayer and the fact that its structure naturally changes in progression across its width (inhomogeneous). Each region of the bilayer contributes to the resistance perceived by a water molecule as it traverse the bilayer barrier, and the water molecule must cross two such leaflets. Resistance values are the inverse of permeability values. Computational models based on the inhomogeneous solubility−diffusion molecule are thus capable of integrating the thermodynamic component and the kinetic component into a “resistance profile”, which can in principle provide a graphical representation of the resistance as a function of distance across the bilayer.49,50 However, the relative contribution of each region (and reason for the value of its contribution) is still a subject of significant debate, and there are few reliable ways to apportion resistance to individual leaflets, let alone regions of one leaflet. But, one key feature keeps reappearing in computational models using the ISDM: the bilayer midplane is a zone of extremely high diffusivity for water molecules. In particular, Orsi has shown that the diffusion coefficient of water is higher in the center of the bilayer than in the outer water phase.51 Shinoda et al.,43 using an explicit-hydrogen model, also reported the diffusion coefficient Dz for water in a DPPC bilayer to be twice as high in the hydrocarbon center than in the water phase. A similar effect seen by Issack and Peslherbe50 leads them to a final resistance profile across the width of the bilayer (taking into account both excess free energy effects and diffusivity): their result is an extremely low level of local resistance in the center of the bilayer, almost as low as the resistance in the water phase. Therefore, if alkane solvent molecules predominantly reside in the bilayer midplane, they can be seen as a third source of resistance to water permeation, in addition to each of the two leaflets and any resistance based on entry into head groups. The bilayer midplane can no longer be considered a free diffusion zone. This effect of hydrocarbon solvent molecules on bilayer water permeability is not believed to have been clearly observed prior to our work. Although it is quite likely that some hydrocarbon solvent has become trapped in the membrane, thus thickening the bilayer, the presence of hydrocarbon molecules may not always be expected to have a straightforward effect. That is because the inclusion of hydrocarbon molecules may result in several factors changing simultaneously, each of which could influence water permeability. Moreover, hydrocarbons themselves differ in important properties, such as viscosity. Given a permeability model in which resistance to water flow depends on both solubility and diffusivity of water in the dense hydrocarbon

demonstrated that the branched DPhPC bilayer were better able to prevent large cavities from forming in their bilayer than were DPPC molecules. This indicated that small penetrants such as water molecules have more restricted motion and a lower rate of diffusion inside branch-chained lipid bilayers. Indeed, water molecules showed lower local diffusion coefficients inside the DPhPC membrane, and this was attributed to the lower mobility and slower dynamics of the branched DPhPC chains.43 It was concluded that chain branching effects on the permeability are related mainly to the reduction of chain dynamics and alteration in chain mobility.43 In recent studies using MD simulations, it has been reported that the branched DPhPC lipid bilayers required a higher critical initial transmembrane potential to break down and to allow a water channel to form due to the bulkiness of the added methyl branches.19 In view of the foregoing, then, the permeability behavior which we have observed for DPhPC may be readily explainable in terms of these computational and other experimental studies.35 Droplet bilayer systems are of necessity performed in the presence of a hydrophobic (oil) solvent, which can become trapped in the bilayer to a greater or lesser extent. Capacitance measurements on DIBs made with DPhPC in various hydrophobic solvents have shown a small but significant difference in bilayer thickness dependent upon the solvent.44 Specifically, the hydrophobic thickness of a DIB formed from DPhPC bilayers in hexadecane is 29.9 Å, whereas this is reduced to 27.8 Å when the solvent is replaced by a mixture of 9:1 silicone oil and hexadecane. In the latter case, the larger average solvent molecules are entropically excluded from the bilayer to a greater extent than the former (although exclusion of larger solvent molecules from the bilayer is often also attributed to the relatively poor solubility of amphiphiles in such solvent57). Thus, it is likely that the hexadecane DIB is not solvent-free. Interestingly, Sarles44 also showed that the presence of 20% cholesterol in the DPhPC DIB in hexadecane had about the same thickness (29.7 Å) as in its absence. Thus, the presence of hexadecane solvent molecules, while clearly occupying volume in the bilayer region, does not thicken the bilayer to any different extent when cholesterol is present relative to the absence of cholesterol. These results can be taken as suggesting a similar presence of hexadecane in our case. Indeed, when we repeated our experiment for the permeability of pure DPhPC but using squalene (a triterpene of formula C30H50), a solvent known to be almost completely excluded from lipid bilayers,45 and compared the permeability values to the C16 case, we found a consistent increase in permeability of ∼30% at all temperatures in the range from 30 to 40 °C. Since our corpus of experiments (for both pure DPhPC and for the S-Chol-containing bilayers conducted below) is conducted in the presence of the same solvent each time (C16), we believe that the influence of hydrocarbon in the bilayer is thus controlled, allowing the relative values to have validity. Nevertheless, it is intriguing that the putative presence of hydrocarbon solvent molecules may have an effect upon water permeability at all, in view of the perspective of some investigators who suggest that the presence of alkane solvent in the bilayer should have little or no effect upon water transport kinetics.46 In fact, there has been little conclusive study to determine the manner in which hydrocarbon solvents may be localized or situated in bilayers, other than to quantify their presence. In studies of lecithin bilayers adjacent to an D

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Figure 4. (a) Osmotic water permeability coefficients (μm/s). (b) Arrhenius plot of symmetric DIB membranes formed from pure DPhPC, and symmetric membranes formed from DPhPC with S-Chol both sides (13 and 44 mol %) at the temperature range of 30−40 °C, and (c) activation energy obtained from the slope of the Arrhenius plot.

core, changes in thickness and fluidity will both influence the permeability. Therefore, despite evidence that bilayers containing hexadecane are thicker than those formed in squalene, they would also comprise solvent of lower visosity, which could promote the solubility and/or diffusivity of water molecules. Water Permeability of Symmetric and Asymmetric DIB Formed from DPhPC with the Addition of S-Chol. We created symmetric DIB membranes from two droplets, each containing vesicles formed of a mixture of DPhPC and S-Chol. We have also created asymmetric DIB membranes from a first droplet containing vesicles formed from only the phospholipid and a second droplet containing vesicles formed of a mixture of DPhPC and S-Chol. Cholesterol sulfate (S-Chol) is included as a component of DPhPC bilayers by codispersion of the sterol in the vesicle solution. In each case, S-Chol at two different concentrations was tested. S-Chol was incorporated at a relative molar ratio to DPhPC from 1:6.3 (13 mol % of S-Chol to total lipids) to 1:1.3 (44 mol % of sterol relative to total lipids). For all experiments, the total concentration of lipids (DPhPC + SChol) was in the regime of 4−5 mM. The creation of lipid asymmetry in the DIB was achieved by incorporating S-Chol in only one side of the droplet but not the other droplet. DIB Made by DPhPC with S-Chol Both Sides (Symmetric DIB). The presence of S-Chol in DPhPC on both sides of DIB membrane reduced Pf for both concentrations of S-Chol (13 and 44 mol %) at all temperatures in the range studied, 30−40 °C, as shown in Figure 4a. The activation energy derived from the slope of the plot in Arrhenius coordinates for DPhPC/SChol membrane was equal to 17 ± 1.0 and 18 ± 0.8 kcal/mol for 13 and 44 mol %, respectively. This is shown in Figures 4b and 4c along with data for pure DPhPC for comparison (Ea of pure DPhPC is 12 ± 0.8 kcal/mol). The Ea for symmetric membranes containing S-Chol was found to be at significantly higher values (∼17−18 kcal/mol) relative to its absence (12 kcal/mol), consistent with a general expected effect for cholesterol and its derivatives upon water transport.52 This effect upon activation energy, however, appears to reach a plateau above a certain S-Chol content between 13 and 44 mol %. That is our observed values for the activation energy for water transport in bilayers containing SChol on both sides shows little noticeable difference regardless of whether the lipid contains 13 or 44 mol % S-Chol; the higher

quantities of sulfated sterol engenders scant further diminution in water transport kinetics. This leveling-off has precedent in MD simulation studies50 for hydrated bilayers of DPPC and cholesterol, where cholesterol was found to have an inhibitory effect on water permeation, yet bilayers containing cholesterol at greater than 20 mol % display a less dramatic dependence on content than at lower concentrations. The investigators in this study attribute the effect of cholesterol in decreasing membrane permeability to a magnification in the free energy barrier to permeation in the dense bilayer interior; that is, cholesterol decreases permeability by lowering the solubility of water in the dense hydrophobic region of the bilayer. Although we do not see a significant decrease in permeability upon moving from 13% S-Chol to 44% S-Chol in symmetric bilayers (i.e., S-Chol in both leaflets), nor a large increase in Ea, we nevertheless discern important differences which appears at higher S-Chol content when we move to discuss asymmetric bilayers, below in this paper. Although the physicochemical properties of S-Chol in biomembranes have not been as thoroughly studied as cholesterol itself, several previous studies have been reported for S-Chol to act as membrane stabilizer and interact with phospholipids in a manner similar to that observed for cholesterol. For example, using fluorescence polarization and differential scanning calorimetry (DSC) techniques, it has been shown that S-Chol suppress the liquid-crystal to gel phase transition temperature of DPPC and increases the acyl chain order, at temperatures above the liquid-crystal to gel transition temperature.53 Similar results have been reported wherein SChol tends to abolish the liquid-crystal-to-gel transition and produced an ordering effect for palmitoyloleoylphosphatidylethanolamine model membranes.29 Faure et al.28 have studied the properties of membranes of dimyristoylphosphatidylcholine (DMPC) with S-Chol, using microscopy, X-ray diffraction, and NMR techniques. They observed that S-Chol has an ordering effect on acyl tails and can promote hydration of DMPC. In addition, S-Chol was shown to stabilize the bilayer organization in phosphatidylethanolamine or phosphatidylethanolamine/ sphingomyelin mixtures. By using DSC, Schofield et al.30 showed that S-Chol decreases the main phase transition temperature and broadens the transitions of model phospholipid membranes including DPPC. Using the molecular E

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Figure 5. Water permeability coefficient for DPhPC with S-Chol in only one side (either water side or osmolyte side) and both sides: (a) S-Chol 13 mol % of DPhPC; (b) S-Chol 44 mol % of DPhPC.

Figure 6. Water permeability coefficient of asymmetric bilayer with S-Chol (a) water side and (b) osmolyte side.

dynamics simulation technique, Smondyrev et al.31 reported an increased chain ordering and a condensing effect at 50 mol % concentrations of S-Chol in DPPC membrane. Our result, that of lower permeability and higher Ea for water permeation at all concentration of S-Chol, is thus essentially consistent with the condensing effect of the acyl chain shown for S-Chol with various phospholipid membranes. DIB Made by DPhPC with S-Chol One Side (Asymmetric DIB). In order to examine effects of the structure of a single leaflet in bilayer on the water permeability, we incorporated SChol (at either 13 or 44 mol % of S-Chol with respect to DPhPC) to only one droplet, either to a droplet containing osmolyte solution or to a pure aqueous droplet. This created an asymmetric DIB, akin to those which were previously described by Bayley.32 It is understood, however, that many sterols are known to flip from one side of a bilayer to the other. For example, in recent studies by Conboy et al.,54 the rate of cholesterol transbilayer migration (flip-flop) was measured by sum-frequency vibrational spectroscopy; they observed that cholesterol redistributes itself from an asymmetric distribution in the bilayer to a symmetric distribution between the two leaflets within about 10 min at 23 °C for 1,2-distearoyl-snglycero-3-phosphocholine bilayer (DSPC). Similar fast motion of cholesterol was observed even at a temperature of 5 °C in the gel phase lipid DSPC.54 But, on the other hand, S-Chol has been reported to exhibit a half-time for flip-flop of at least 14 h (ca. 50 000 s).33,55 This slow transbilayer movement has been

explained to be due to the increased energy required to move charged sterols (S-Chol contains a negatively charged sulfate moiety) from the inner to outer leaflet of the bilayer through the hydrophobic interior of the membrane. The effects of the large negatively charged head group possessed by S-Chol has been long been known as a feature of significant contrast to cholesterol.19 Since the rate of flip-flop for S-Chol is clearly far slower than our experimental time scale (the latter being no more than about 600 s), we believe that the bilayer asymmetry created by S-Chol in the DIB is maintained during our permeability measurement. As shown in Figure 5a, an asymmetric bilayer containing 13 mol % of S-Chol in water droplet side decreased Pf from its original value of 42 μm/s for pure DPhPC at 30 °C to 33 μm/s. Recall that the permeability coefficient for having S-Chol of 13 mol % on both sides of a DPhPC membrane (symmetric DPhPC/S-Chol) is 18 μm/s at 30 °C. Note that the permeability value of 33 μm/s for asymmetric DPhPC/SChol at 13 mol % falls between the value obtained with pure DPhPC and symmetric DPhPC/S-Chol bilayers. Figure 5a also shows asymmetric bilayer having S-Chol only in the osmolyte droplet side instead of the water droplet side. A similar permeability value of 34 μm/s at 30 °C was obtained, and a comparable corresponding reduction in permeability was observed at 35 and 40 °C as well. It appears that no noticeable difference was observed whether S-Chol is located either in pure water droplet or in osmolyte droplet. That is, even given F

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Langmuir that water flow in an osmotically imbalanced system is always from the pure water droplet to the salty droplet, it mattered little insofar as Pf values were concerned whether the S-Cholcontaining leaflet was proximate to the water-egress droplet or to the water-ingress droplet. Figure 5b shows the permeability values for a relatively higher concentration of S-Chol, 44 mol % with respect to phospholipid. When S-Chol is present at this concentration on one side of the DIB (asymmetric), water permeability decreased further to 24 μm/s at 30 °C for both cases when S-Chol is in the water side or the osmolyte side. Once again, this value is between the value obtained with pure DPhPC (42 μm/s) and symmetric DPhPC/S-Chol bilayers (16 μm/s) at 30 °C, as it was the case with the S-Chol concentration at 13 mol %. For these asymmetric bilayers, we can also discern a marked dependence on S-Chol concentration for permeability values in the presence of sterol in the relevant leaflet. As shown in Figure 6, the Pf value at 30 °C was reduced (relative to pure DPhPC) to a value of 33 μm/s for 13 mol % S-Chol in one leaflet and further reduced to 24 μm/s for 44 mol % in that leaflet. Analogous behavior was seen for 35 and 40 °C. Similar results were seen for the condition of the sterol-containing leaflet being on the side of the pure water droplet and the osmolyte droplet side. Therefore, when viewed from the vantage point of permeability coefficient, added sterol to one side would enhance the resistance of the bilayer to the water permeation process. This is consistent with a model wherein the progressive addition of S-Chol to one DPhPC leaflet brings about a correspondingly progressive reduction in free volume in the dense hydrocarbon core of the acyl tails of the DPhPC of the affected leaflet, while leaving the opposite leaflet unaffected. The overall observed permeability, being the reciprocal of the summation of the instantaneous resistances “felt” by a water molecule as it traverses the bilayer, would begin to precipitously drop upon S-Chol addition to a single leaflet, and a continued drop in Pf would be visible for further S-Chol addition to one side. However, from delivery of S-Chol to both sides (symmetric bilayer), free volume in the hydrophobic core regions of the bilayer as a whole is naturally more rapidly reduced. We have also determined the activation energy by measuring the temperature dependence of the water permeability. The mean activation energies obtained for our asymmetric DIB were 14−15 kcal/mol, regardless of whether the S-Chol is presented on the osmolyte side of the bilayer or pure-water side (Figure 7). Our results appear to indicate that a compositional change in even a single leaflet of bilayer can have an influence on its water permeability value and thus capability of regulating water transport. Similar findings were reported by Zeidel et al.,55 who reported the permeability of water across artificial symmetric and asymmetric planar bilayers, formed from DPhPC with 25% w/w S-Chol. It is important to note, however, that our observed effect relating to independent resistances for individual leaflets does not clearly evidence itself unless the leaflet contains a sufficiently high level of S-Chol in addition to the DPhPC. That is, the respective leaflets ought to have significant differences in their permeability behavior, or else the effect does not manifest itself. The principle for this conclusion, as discussed by Krylov8 and Negrete,55 is that the overall resistance to water permeation (resistance being the inverse

Figure 7. Comparison of mean activation energy (kcal/mol) for pure DPhPC, DPhPC with S-Chol one side (either water side or osmolyte side), and DPhPC with S-Chol (both side). S-Chol, when present, was at two differing values (13 and 44 mol %).

of permeability Pf) is the sum of the inverse of the resistance offered by each leaflet, shown in eq 2: 1/Pf = 1/Pa + 1/Pb

(2)

where Pa is the permeability of the first leaflet and Pb is the permeability of the second leaflet. When we employ the data from Figure 6 for the case wherein the leaflet containing S-Chol at 44 mol % of total lipids, we find, as shown in Table 1, that the predicted net permeability is a Table 1. Calculated vs Observed Permeability for Asymmetric Bilayer Containing 44 mol % S-Chol in a Single Leaflet calcd net permeability based on sum of individual resistances (μm/s) (eq 2)

obsd net permeability (μm/s)

arithmetic mean (μm/s)

temp (°C)

23 34 55

24−25 37−39 52−55

29 39.5 61

30 35 40

remarkably close approximation to the observed net permeability (across both bilayers), one that appears to be significantly better explained by being the sum of individual resistances rather than being the arithmetic mean of the respective permeabilities. However, for the case where the sterol-containing leaflet is formulated to contain only 13 mol % S-Chol (not shown in Table 1), the observed overall permeabilities are better explained by the arithmetic mean of permeabilities for the SChol being in both leaflets (symmetric) and no sterol. This result can be taken to indicate that there needs to be sufficient differentiation in the water permeation resistance between leaflets before one can observe them acting as independent resistors. Moreover, the fact that only the asymmetric bilayer having S-Chol at 44 mol % on the sterol side shows this behavior would tend to imply that neither is the effect of SChol saturating at a value below 44 mol % nor is S-Chol being depleted from the bilayer by a hypothetical dissolution into the oil phase; were its effect to be saturated or it to be depleted would obviate the possibility that the high levels of S-Chol would have a different behavior than the lower levels. Other experimental findings for permeability across an asymmetric planar membrane have confirmed that leaflets in G

DOI: 10.1021/acs.langmuir.5b02748 Langmuir XXXX, XXX, XXX−XXX

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Langmuir

demonstrate that a sterol-containing leaflet can act as an independent resistor vis-á-vis the phospholipid leaflet not including S-Chol, but only if the concentration of S-Chol is sufficiently high. Such a sterol concentration study for asymmetric bilayers is not believed to have been performed previously. This result can be taken to indicate that there needs to be sufficient differentiation in the water permeation resistance in situations of sterol asymmetry before one can observe them acting as independent resistors. This insight pertaining to the study of sterol asymmetry is important for understanding of its profound effects in neurological cellular systems. We also analyzed the effect of hydrocarbon solvent, included in the bilayer from the hydrophobic oil phase, upon water permeability. The permeability of pure DPhPC in squalene, a solvent known to be almost completely excluded from lipid bilayers, exhibits a consistent increase in value of ∼30% at all temperatures in the range from 30 to 40 °C. This result contrasts with the perspective of investigators who have suggested that the presence of alkane solvent in the bilayer should have little or no effect upon water transport kinetic. If alkane solvent molecules predominantly reside in the bilayer midplane, as suggested by prior theoretical and experimental studies, then these molecules can be seen as a source of resistance to water permeation, in addition to each of the two leaflets and any resistance based on entry into head groups. This effect of hydrocarbon solvent molecules on bilayer water permeability is not believed to have been clearly observed prior to our work. The engineering of our various systems is believed to have implications for future studies in which detailed knowledge regarding the transport (both passive and active) of small moieties (neutral and ionic) across bilayers is needed at both the cytoplasmic and exofacial levels. In addition, our findings highlight an insight that differences in membrane sterol content combined with variations in temperature can be used as an effective way of controlling membrane water permeability, important for the study of model membranes of a wide variety of cellular types in living systems.

a bilayer can offer independent and additive resistances to water permeation.8 These results also suggested that the resistance to permeation of a bilayer is the sum of the resistances to permeation of its two leaflets and concluded that a single leaflet of a membrane bilayer can act as independent barrier to permeation in an asymmetric bilayer. Additionally, a nuclear magnetic resonance (NMR) study of the thermal behavior of small single-walled vesicles, composed of DMPC, has shown that the two halves of the bilayer are so weakly coupled that they can undergo phase transitions independently (gel to liquid-crystalline), indicating that individual leaflets of a membrane could alter their physical properties without influence from the other leaflet.12 However, more recent MD studies on interleaflet interaction in membranes hint at the existence of various types of interleaflet coupling within a bilayer,13 such as induced curvature and associated changes in lipid order being one source of interleaflet coupling in asymmetric bilayers. Sterol asymmetry is a specific case of membrane asymmetry and can have profound effects in neurological cellular systems. It has been found in studies of wide variety of mammals that the cytofacial leaflet of the synaptic plasma membrane (SPM) contains over 85% of the total SPM cholesterol vs the exofacial leaflet. As a consequence, the SPM cytofacial leaflet has less fluidity than the exofacial leaflet, which influences the ability of molecules to partition into the SPM. The asymmetry, while ordinarily tightly regulated, is seen as being modifiable by aging and by statin and ethanol intake. Changes in cholesterol asymmetry are believed to have effects upon fluidity, lipid domains changes, transbilayer diffusion, and protein function.10,56 While it is beyond the scope of our paper to discuss the details of the nature of interleaflet coupling in asymmetric bilayers, our results can be understood to add to the pool of evidence that individual leaflets of membrane bilayers could have altered physical properties independent of the other leaflet.

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CONCLUSION Membrane researchers have long enjoyed the high bilayer stability and unique structural and dynamic properties of DPhPC membranes, for studying electrophysiological properties and various biological membrane processes, such as the interaction between lipid bilayers and proteins, peptides, and other biological molecules. In light of this, we have investigated the water permeability behavior of pure DPhPC droplet bilayer membranes as well as of symmetric and asymmetric DIBs formed by DPhPC and S-Chol at 13 and 44 mol %. The permeability coefficients (Pf) obtained at 30, 35, and 40 °C were 42 ± 3, 54 ± 4, and 80 ± 6 μm/s, respectively, corresponding to an activation energy of 12.1 ± 0.8 kcal/mol. The presence of S-Chol in DPhPC on both sides of droplets DIB (symmetric DIB) reduced Pf at both concentrations (13 and 44 mol %) and increased the activation energy (Ea) to 17− 18 kcal/mol. When only one side of the DIB contains S-Chol (asymmetric DIB), an Ea of 14−15 kcal/mol was obtained, a value intermediate that of pure and symmetrical DIB containing S-Chol. In this paper, we have demonstrated that compositional changes to individual leaflets can be achieved by the droplet bilayer method and their effects manifested by observable changes to the water permeation process. The data are consistent with a capability for regulation of water transport by one leaflet independent of the other. Our results

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02748. Description of the overall method for determining permeability measurement using DIB (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel 914-633-2638; Fax 914-633-2240; e-mail [email protected] (S.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Science Foundation (NSF-CHE-1212967). P.J.M, M.M, and J.D. thank the Patrick J. Martin Foundation for Scholarship.

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