Fluidity Modulation of Phospholipid Bilayers by Electrolyte Ions

Article pubs.acs.org/JPCA

Fluidity Modulation of Phospholipid Bilayers by Electrolyte Ions: Insights from Fluorescence Microscopy and Microslit Electrokinetic Experiments Ralf Zimmermann,*,† David Küttner,† Lars Renner,† Martin Kaufmann,† and Carsten Werner†,‡ †

Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Hohe Strasse 6, 01069 Dresden, Germany ‡ Technische Universität Dresden, Center of Regenerative Therapies Dresden, Tatzberg 47, 01307 Dresden, Germany S Supporting Information *

ABSTRACT: Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH− ≫ H3O+). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.

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INTRODUCTION Cell membranes separate the intracellular from the extracellular space and regulate important cellular processes. They primarily consist of lipids, proteins, and sugars.1 Corresponding to the variety of cell types and cellular functions, a huge variety of these components can be found in cell membranes. Membrane lipids vary in chain length, chain saturation, and headgroup structure. About 90% of the lipids in biomembranes are zwitterionic.2 Phosphatidylcholines (PCs) represent with approximately 50% the major fraction.2 Therefore, the exploration of structural and dynamical properties of membranes composed of PC is of large interest for different fields of fundamental and applied research.3−10 A key property of lipid membranes is their phase behavior. In general, lipid membranes can exist in different phases, e.g., the liquid crystalline or fluid phase, gel phase, subgel phase, and ripple phase.4,5 Often, the phase behavior is determined by the gel (Lß)−fluid (Lα) transition.5 The phase transition temperatures primarily depend on the composition of the membrane and the structure of the lipids. As lipid headgroups consist of various acidic and/or basics moieties, the majority of lipids is charged in aqueous environments. The resulting electrostatic interactions within the lipid membranes as well as between lipid headgroups and ions from solution are crucial for phase transition temperatures,6 membrane fusion,7 and transmembrane transport.8 © 2012 American Chemical Society

The influence of charge on membrane properties was often discussed in terms of the Gouy−Chapman mean-field theory that predicts equal behavior for ions with the same valency.9 During the past decade, an increased number of studies were dedicated to the investigation of ion-specific phenomena at lipid bilayer membranes. For that purpose, fluorescence correlation spectroscopy,10−12 fluorescence solvent relaxation,11 membrane dipole potential measurements,13,14 molecular dynamics (MD) simulations,10,15−18 and charge displacement techniques19 have been applied. Measurements at zwitterionic lipid membranes have shown that several ions are penetrating into the bilayer, that some are adsorbing at the liquid/ membrane interface, and that others do not even attach.10,11,14 The reasons for the partially contradictory results are still under debate. Concepts of van der Waals forces that mediate interactions of lipids with the ions at the interface as well as kosmotropic (structure forming) effects and water affinities have been taken into account. In some of the aforementioned studies,11,14,19 it was assumed that the acidic and basic moieties in zwitterionic lipid headgroups bear an equal and opposite charge in the neutral pH range, and both charges compensate each other (no net charge). In contrast, recent electrophoretic Special Issue: Herman P. van Leeuwen Festschrift Received: December 22, 2011 Revised: February 3, 2012 Published: February 3, 2012 6519

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times with ultrapure water. Subsequently, the desired electrolyte solution was injected. The formation of homogeneous and stable lipid membranes was confirmed by fluorescence microscopy. In line with results of X-ray diffraction measurements22 and MD simulations,23 the average lipid area in the DOPC membranes was found to be (0.72 ± 0.02) nm2 by ATR-FTIR spectroscopy.20 Fluorescence Recovery after Photobleaching (FRAP). Diffusion coefficients and mobile fractions were determined by FRAP experiments24 on a fluorescence confocal laser scanning microscope TCS SP5 (Leica, Bensheim, Germany). In short, a defined spot with a diameter of (10 ± 1) μm was bleached in the bilayer using a short (86 ms) high-power laser beam. The recovery kinetics were recorded with a 63× objective (Leica, NA = 1.4) at 128 × 128 pixels with a delay of 86 ms between each image. The diffusion coefficients were calculated following the approach of Soumpasis.25 The mobile fraction of lipids within the photobleached area was estimated from the fluorescence intensities as described elsewhere.20 Streaming Current Measurements. Streaming current measurements were performed at rectangular streaming channels formed by two sample surfaces (length, 20 mm; width, 10 mm; separation distance, 30 μm) using the Microslit Electrokinetic Set-up.26,27 Zeta potentials (ζ) were calculated from the streaming current data according to the Smoluchowski equation.28 The isoelectric points were derived from the plot of the zeta potential vs pH at ζ(pH) = 0. The measurements were started in the alkaline pH range. Each pH was equilibrated for approximately 40 min. Electrolyte Solutions. Electrolyte solutions of different ionic strength were prepared from deionized and degassed water (Milli-Q Gradient A10, Millipore Corp.) by addition of 0.1 M stock solutions of the corresponding salts. The solution pH was changed by the addition of 0.1 M HCl and KOH. To eliminate the influence of the ion valence on the extension of the diffuse layer (Debye length), the pH-dependent measurements with the different electrolytes were performed at similar ionic strength. Please note that this approach is related to different concentration levels for monovalent and bivalent ions.

and streaming currents measurements revealed a significant negative charge at neutral pH and isoelectric points of about 4 for zwitterionic lipid membranes.13,20 The sign reversal of the membrane charge to positive values at pH ∼ 4 was found to be correlated with a decrease of the area compressibility modulus and a reduction of the lipid mobility, respectively. The variation of the membrane properties was attributed to a charge-induced transition of the fluid bilayer in a gel/ordered phase bilayer. In this study, we systematically applied fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements to study correlations between the fluidity and net charge of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) membranes in the presence of various kosmotropic and chaotropic ions. Measurements were performed at varying pH and ionic strength of NH4Cl, KCl, NaCl, LiCl, CaCl2, MgCl2, KBr, KNO3, KI, and KClO4 solutions. Furthermore, for KCl, KClO4, and MgCl2, diffusion coefficients were determined at varied temperature. The data indicate that ion−lipid interactions superimpose the primary charge formation processes and influence the fluidity of the supported bilayer lipid membranes (sBLMs) at different temperatures. On the basis of the experimental results, we discuss the impact of the different electrolyte ions on the lipid charge, conformation, and hydration. Furthermore, we provide an explanation for the observed effects taking into account results from molecular-scale experiments and simulations.

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MATERIALS AND METHODS Substrates. For all FRAP experiments, sBLMs were prepared on coverslips (Corning B.V. Life Sciences, Netherlands). Home-built glass cylinders were glued (NuSil, California, USA) on top of these substrates. The total applicable solution volume of the assembly was 300 μL. The electrokinetic measurements were performed on sBLMs prepared on top of thermally oxidized silicon wafers (30 nm oxide layer). The substrates were cleaned in a mixture of ammonia hydroxide (29 wt %, Acros Organics, Geel, Belgium), hydrogen peroxide (35 wt %, not stabilized, Merck, Darmstadt, Germany), and deionized water (NH4OH:H2O2:H2O = 1:1:5) at 70 °C for 10 min and rinsed intensively in deionized water. Prior to the preparation of DOPC membranes, the substrates were treated in a plasma chamber at high RF for 2 min to render the surface hydrophilic (Harrick Plasma, Ithaca, USA). Lipid Membrane Preparation. DOPC membranes were prepared according to the protocols described in detail elsewhere.20 In short, DOPC was dissolved in chloroform to give a final concentration of 5 mg mL−1. After evaporating the solvent, the lipid was hydrated in pH 4 saline (10 mM NaCl, 5 mM CaCl2, adjusted to pH 4 with 0.1 M HCl) and extruded (Mini Extruder, Avanti Polar Lipids, Alabama, USA) 31 times through a polycarbonate membrane (pore size, 50 nm, Whatman Ltd., UK) following the procedure of Hope et al.21 For FRAP measurements, the fluorescent probe 1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) was additionally added at a concentration of 1 mol % to the DOPC in chloroform. DOPC and NBD-PE were purchased from Avanti Polar Lipids (Alabama, USA). All other chemicals were purchased from Sigma Aldrich (Munich, Germany) and used without further purification. For the chemical structure of DOPC and NBD-PE, see ref 20. The lipid vesicle solution (0.2 mg mL−1 in 10−3 M KCl solution) was injected into the assembled measuring cells. After 2 h incubation at 22 °C, the cells were excessively rinsed 10

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RESULTS FRAP Measurements at Room Temperature (22 °C). In Figure 1a, we show the pH dependence of the diffusion coefficient of the DOPC membranes in 1 mM (ionic strength) KCl, KClO4, and MgCl2 solutions. Similar to our previous study,20 a rather constant mobility of (6 ± 1) μm2 s−1 in the pH range from 9 to 6, a slight decrease at pH 4 to ∼4.5 μm2 s−1, and a significant drop in mobility to about 1 μm2 s−1 at pH 2 were found in KCl solution. Measurements in 0.1 mM and 10 mM KCl solution (data not shown) revealed that the observed behavior is rather independent of the ionic strength. The replacement of the Cl− by the ClO4− ion reduced the effect of the pH on the lipid mobility at acidic pH (Figure 1a), while similar mobilities were determined in the neutral and alkaline pH range. In a series of measurements in 1 mM electrolytes with anions of different charge to size ratio, stronger effects were found with increasing chaotropicity (decreasing free hydration energy)29 of the anion according to the following series: Cl− ≈ Br− < NO3− < I− < ClO4− (Figures 2 and S1 in Supporting Information). The influence of cations on the membrane fluidity was studied using NH4Cl, NaCl, LiCl, CaCl2, and MgCl2 solutions. The lipid mobility remained rather unaffected (compared to 6520

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observed only a slightly increased lipid mobility in the weak acidic pH range (pH 3−4). Rather similar lipid mobilities were also found at neutral and alkaline pH in CaCl2 and MgCl2 solutions. Below pH 6 FRAP revealed higher lipid mobilities in the presence of the Ca2+ and Mg2+ ions. In a series of additional measurements at ionic strength of 10 mM and 100 mM, we observed an increasing mobility with increasing Ca2+/Mg2+ concentration at acidic pH, while the lipid mobility remained rather unaffected at neutral and alkaline pH (Figure S2, Supporting Information). At pH = 2 the Mg2+ ions showed the strongest influence on the lipid mobility, while at the higher pH values the effect was more pronounced for the Ca2+ ions. Furthermore, the mobile fraction was found to be higher than 90% for all electrolyte compositions. FRAP Measurements at Varying Temperatures. To unravel the fundamental processes causing the variation of the membrane fluidity in the acidic pH range, we performed additional measurements at varying temperatures in 1 mM (ionic strength) KCl, KClO4, and MgCl2 solutions. Irrespective of the electrolyte, the diffusion remained rather high in the entire pH range at 45 °C (Figure 1b). Decreasing the temperature of the electrolyte at pH 2 correlated with a drop of the lipid mobility (Figure 1c). The transitions from the high to the low lipid mobility occurred within a rather broad temperature range characteristic for the electrolyte. Compared to KCl, the transitions occurred within a smaller temperature range and lower temperatures in the KClO4 and MgCl2 solutions. Furthermore, the end points of the cooling curves were similar to the values determined in the pH-dependent experiments at 22 °C (Figure 1a). Streaming Current Measurements. Streaming current measurements revealed a strong influence of the pH and ionic composition of the electrolyte on the charging of the DOPC membranes. In Figure 3, we present the isoelectric points as a function of the free energy of ion hydration for the different electrolyte ions and ionic strength used for the electrokinetic measurements. In our previous study,20 the DOPC membranes showed negative zeta potentials in the neutral and alkaline pH range and an isoelectric point of 3.9 ± 1 in KCl solutions of different ionic strength. In Figure 3, we show this IEP as a reference. Compared to the rather invariant IEPs in KCl solutions of different ionic strength, variation of the anion composition of the electrolyte revealed significant variations of the IEP to lower pH values (Figure 3). The shift of the IEP was found to be more pronounced for the more chaotropic anions29 and increased with increasing salt concentration. Similar IEPs as in KCl were also determined for the DOPC membranes in NH4+, Na+, and Li+ containing chloride solutions (Figure 3). Independent of the ionic strength the IEPs were found in the range 3.9 ± 0.2. In contrast, the presence of various concentrations of the divalent cations Ca2+ and Mg2+ caused a significant shift of the IEP to higher pH values (Figure 3). The IEPs were detected between pH 4.0 and pH 5.4 in CaCl2 and pH 3.9 and pH 6.5 in MgCl2 solutions. Altogether, the zwitterionic DOPC membranes showed a negative excess charge at neutral pH and isoelectric points of about 3.9 in KCl solution. The IEP of the DOPC membranes was shifted to lower values in solutions with increasing chaotropicity of the anions, whereas higher IEPs were observed in the presence of kosmotropic cations. The effects are more pronounced at higher solution concentrations for the more chaotropic anions and kosmotropic cations, respectively.

Figure 1. Diffusion coefficients of sBLMs prepared from DOPC doped with 1 mol % NBD-PE in KCl, KClO4, and MgCl2 solutions (I = 1 mM) of varied pH at 22 °C (a) and 45 °C (b) as well as at varied temperature at pH 2 (c). In (a) the black arrows indicate the tendencies observed for the shift of the IEP in electrolytes containing various types of cations and anions. The observed variation of the diffusion coefficient under similar conditions is indicated by the gray arrows.

KCl) in the presence of the NH4+, Na+, and Li+ ions (see Figure 2 and S1, Supporting Information). For the Li+ ions we 6521

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Figure 2. Diffusion coefficients of sBLMs prepared from DOPC doped with 1 mol % NBD-PE for different solution pH in the presence of 1 mM electrolyte solution (ionic strength) of varied anionic (left) and cationic (right) composition. Ions are classified according to their Gibb's free energies of hydration ΔGhyd. The color of the data points corresponds to the pH values given in the middle of the figure. All measurements were performed at 22 °C.

significant negative membrane charge at neutral and alkaline pH and, rather independent of the ionic strength, a positively charged membrane below the IEP of 3.9 ± 0.1. We attribute the negative membrane charge above the IEP to the unsymmetrical adsorption of water ions,30−32 i.e., the fact that OH− ions are stronger adsorbed as compared to H3O+ ions. At pH values below the IEP, the adsorption of H3O+ ions dominates over the adsorption of OH− ions at surfaces without ionizable groups.32 In the case of the DOPC membranes, we consider the increasing surface concentration of H3O+ ions in the acidic pH range to be accompanied by a protonation of the phosphate group of the lipids. As the IEP was almost identical at all KCl solution concentrations, we exclude significant adsorption of K+ and Cl− ions at the concentration levels used in this study. The shift of the IEP into the more acidic pH range with increasing chaotropicity of the anions (Figure 3) clearly indicates that anions with a weakly bound hydration shell show a higher tendency to interact with lipid membranes. By contrast, the streaming current measurements revealed significant shifts of the IEP to values higher than 3.9 for the multivalent cations according to their Gibbs free energy of hydration (Figure 3). No effects were observed for the monovalent cations. Neither the chaotropic NH4+ ion nor the kosmotropic Li+ ion caused a significant change of the IEP. Charge displacement experiments19 and dipole potential measurements33 revealed noticeable differences in the interaction of monovalent cations with lipid vesicles and membranes at higher salt concentrations. However, because of the decreasing sensitivity of streaming current measurements to small differences in electrosurface characteristics at high ionic strength, we did not perform streaming current measurements above an ionic strength of 10 mM. Beyond the tendency of ions to interact with lipid molecules, the preferred adsorption site in the bilayer membrane depends on the structure of their hydration shell17,18 and thus influences the pattern of charge formation and compensation. The small Cl− ion with its tightly bound water molecules (compared to larger anions) was found to be weakly adsorbed at salt

Figure 3. Isoelectric points of sBLMs prepared from DOPC on SiO2 substrates in the presence of different anions and cations at a temperature of 22 °C. Ions are classified according to their Gibb's free energies of hydration ΔGhyd. The horizontal dotted line indicates the IEP of 3.9 obtained in 1 mM KCl solution.

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DISCUSSION

The results presented above revealed a significant influence of the pH and ionic composition of the electrolyte on the fluidity and charging of zwitterionic DOPC membranes. Below, we start the discussion with the interpretation of the results from the streaming current measurements. Following that, the impact of the different electrolyte ions on the lipid conformation and membrane fluidity is discussed. Charging. The DOPC headgroup contains both an acidic and a basic moiety that should charge up equally in opposite directions at neutral pH. Therefore, considering the molecular structure only, one would expect an electrically neutral membrane in the intermediate pH range. By contrast, streaming current measurements performed in KCl solutions revealed a 6522

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study for the preparation of the zwitterionic lipid membranes are negatively charged due to deprotonation of the hydroxyl groups (IEP ∼ 2).20 As OH− and H3O+ ions are able to penetrate phospholipid membranes without the existence of membrane defects,38 we imply similar charging of the inner and outer leaflet of the DOPC membranes. Under these conditions, the positive net charge below pH 3.9 would be related to an increasing attraction between the lipids in the inner leaflet of the membrane and the substrate and therefore to a reduced lipid mobility. The higher lipid mobility found at acidic pH in the presence of chaotropic anions (Figures 1a and 2) correlates well with the shift of the IEP in this direction. As discussed above for the water ions, binding of these ions to the lipid membrane increases the lateral electrostatic pressure and thus shifts the transition pH into the more acidic range. In line with our findings, Aroti et al.39 reported an increase in the area per lipid headgroup after the association of hydrophobic anions. Furthermore, similar to water ions,38 hydrophobic anions can penetrate lipid membranes without the existence of membrane defects.40 Binding of these ions to the inner leaflet of the lipid membrane would be related to an extension of the pH range where electrostatic repulsion occurs between the substrate and the sBLM and thus support the fluid state. In addition, binding of anions in the hydrophobic region facilitates the in-plane orientation of the lipid headgroup17 that is less favorable for dense lipid packing and therefore for a transition into the gel phase. In line with the discussed mechanisms, we observed stronger effects with increasing salt concentration. At higher concentrations than used in this study, the chaotropic anions can cause opposite effects due to the disruption of the structure of membrane bound water and the related decrease in the repulsive hydration pressure.41 The weak interactions of the monovalent cations with the DOPC membranes observed in the streaming current measurements were also reflected by the FRAP experiments. The pH dependence of the lipid mobility (Figures 2 and S1, Supporting Information) was similar in 1 mM NH4Cl, KCl, NaCl, and LiCl solution. Therefore, we exclude a strong interaction of these ions with the zwitterionic DOPC at concentrations below 10 mM. The rather unaltered lipid mobilities found at neutral and alkaline pH upon replacement of the KCl by MgCl2 and CaCl2 solutions well agree with results of other studies at similar salt concentration.42 For higher concentrations, numerous authors report increased phase transition temperatures and rigidification of zwitterionic bilayer lipid membranes.35,43,44 The increase of the transition temperature in the presence of multivalent cations is mainly attributed to two effects: formation of ion−lipid complexes and dehydration of the lipid headgroups.35,44 Although the data of our electrokinetic experiments clearly indicate binding of Ca2+ and Mg2+ ions (Figure 3) to the DOPC membranes, the FRAP measurements do not provide any hint on a reduction of the lipid mobility. In contrast, Ca2+ and Mg2+ ions even support unhindered lipid diffusion at low pH as observed for chaotropic anions. At the ionic strength of 100 mM, the bilayers remained completely fluid over the whole pH range (Figure S2, Supporting Information), suggesting phase transition temperatures below 22 °C under these conditions. As no straightforward explanation of the observed cation effect is obvious from the experimental data, further investigations with molecular scale resolution are required. Subsequently, we discuss some facts

concentrations above 0.25 M at the choline group, while chaotropic (more hydrophobic) anions can shed bound water molecules more easily and penetrate deeper into the bilayer interior.17,18 The latter are preferentially adsorbed in the carbonyl region of the lipid.17,18 For the cations, 2H NMR measurements34 and MD simulations35,36 revealed preferred binding to the phosphate group of PC lipids. In line with their higher valence, adsorption of multivalent cations to the phosphate group caused significant variations of the membrane charge (IEP) already at rather low ion concentrations in solution. Less information is available on the position of adsorbed water ions within the bilayer. Both the tendency of OH− ions to assemble at hydrophobic surfaces of various natures and their rather low hydration energy (compared to multivalent cations) suggest that these ions are preferentially adsorbed at the hydrophobic region of the bilayer.13 As discussed above, in the acidic pH range the preferentially adsorbed H3O+ ions most likely bind at the phosphate group (which would be equivalent to a protonation). In summary, the results of our electrokinetic study suggest that the charging of the zwitterionic DOPC membranes is superimposed by the ionization of the phosphate and choline moieties in the lipid headgroup and binding of ions to the lipid membrane. In electrolytes containing neither chaotropic nor kosmotropic ions, unsymmetrical adsorption of water ions (OH− ≫ H3O+) significantly contributes to the overall membrane charge. Additionally, charging is superimposed by the accumulation of chaotropic anions and kosmotropic cations in the headgroup region. Comparison of the salt concentrations that are required to shift the IEP to values lower or higher than 3.9 with the small concentrations of OH− and H3O+ ions in solution that cause strong variations of the zeta potential in the broad pH range (Figure S3, Supporting Information) confirms the dominating role of unsymmetrical water ion adsorption for the charging of zwitterionic lipid membranes at ionic strength below 10 mM. Fluidity. The sign reversal of the membrane charge at pH 4 in KCl solutions was found to be accompanied by a significantly reduced lipid mobility in the acidic pH range suggesting a transition from a fluid bilayer at neutral and alkaline pH into a gel/ordered phase bilayer at acidic pH. Obviously, the unsymmetrical adsorption of OH− and H3O+ ions modulates the balance between attractive and repulsive forces in the bilayer as well as between the bilayer and the substrate and thus the phase transition temperature. The mechanisms underlying the observed transition are not directly obvious from the experiments. We consider the interplay of different effects. (i) Adsorbed electrolyte ions can change the orientation and hydration of the lipid headgroups.17,37 Therefore, it is reasonable to assume that adsorbed water ions also induce similar changes. Simulations of DOPC bilayer membranes in the absence of adsorbed ions37 revealed that the mean value of the tilt angle distribution is ∼78°; i.e., the lipid headgroup is aligned almost parallel to the bilayer plane. A positive excess charge due to adsorbed H3O+ ions in the bilayer interior would cause a stronger orientation of the choline group toward the liquid phase (decrease of the tilt angle) and allow to increase the lipid packing along with a transition of the bilayer in the gel/ordered phase. Furthermore, increasing protonation of the phosphate group due to the excess of adsorbed H3O+ ions below pH 4 will be accompanied by a reduction of the headgroup hydration and therefore induce a shift to higher transition temperatures. (ii) The SiO2 substrates used in this 6523

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CONCLUSIONS In the present work, we systematically studied the influence of specifically binding ions on the fluidity and charging of sBLMs prepared from zwitterionic DOPC. It was demonstrated that unsymmetrical adsorption of water ions (OH− ≫ H3O+) with characteristic isoelectric points around pH 3.9 determines the charging of the membranes in solutions containing neither kosmotropic nor chaotropic ions. Under these conditions, the sign reversal of the membrane charge to positive values was found to be correlated with a transition from a liquid-crystalline bilayer (pH > IEP) into an ordered/gel phase bilayer (pH < IEP). Taking this data as a reference, we demonstrated how kosmotropic and chaotropic ions superimpose the charging by unsymmetrical water ion adsorption and thus influence the fluidity of the bilayer. The IEP of the DOPC membranes was shifted into the acidic pH range in solutions containing chaotropic anions, whereas higher IEPs were observed in the presence of kosmotropic cations. The effects were found to be more pronounced at higher solution concentrations for the more chaotropic anions and kosmotropic cations, respectively. Despite their different impact on the charging of the DOPC membranes, chaotropic anions and kosmotropic cations similarly influence the fluidity of the bilayer in the acidic pH range. As compared to FRAP measurements in KCl solutions, both ion types caused higher lipid mobilities in the acidic pH range suggesting a significantly decreased phase transition temperature under these conditions. On the basis of data available from NMR experiments and MD simulations, we attributed the observed effects to the adsorption of the ions at polar sites in the lipid headgroup region and to the resulting variations of the headgroup orientation and lipid packaging density.

that we consider relevant in this context: (i) The number of ions that bind to lipid membranes and the binding stoichiometry strongly depend on the experimental conditions. The association constants reported for the binding of Ca2+/ Mg2+ ions at PC membranes scatter between 0.1 and 450 M−1.45,46 The majority of experimental data reported in the literature is best described with the formation of ternary complexes between one Ca2+/Mg2+ ion and two phospholipids.45 If we imply an intermediate binding constant of 10 M−1 for our system, estimation of the ion to lipid ratio according to a Langmuir binding model46 reveals that less than 1% of the lipids is involved in the complex formation with Ca2+/Mg2+ ions around the IEP. (ii) In the acidic pH range, the number of binding sites for Ca2+/Mg2+ ions decreases due to the protonation of the phosphate group (see above). Furthermore, the effective concentration of the bivalent cations near the membrane is smaller than the bulk concentration because of the positive membrane charge. While both effects reduce the number of ions that bind to the lipid membrane, the lipid mobility increased in the acidic pH range in the presence of Ca2+/Mg2+ ions. (iii) The 1:2 binding stoichiometry mentioned above was derived under conditions where the headgroups of the zwitterionic lipids are completely ionized. As the density of lipids with ionized phosphate groups decreases in the acidic pH range due to protonation, it is reasonable to assume that the binding stoichiometry changes from 1:2 to 1:1 under those conditions. This process in turn can cause variations of the lipid conformation as well as reorganization of water in the headgroup region. In this context, Binder and Zschörnig47 reported some interesting insights into the hydration of zwitterionic lipid membranes in the presence of Ca2+ and Mg2+ ions. From the variation of the IR adsorption bands, they concluded on the partial dehydration, a conformational change, and immobilization of the phosphate groups. At the same time, they found a complex rearrangement in the carbonyl region, which involves conformational changes and hydration.47 The latter could be one of the reasons for the unhindered lipid diffusion in the acidic pH range. The almost invariant lipid mobilities determined at 45 °C in 1 mM KClO4, KCl, and MgCl2 solution (Figure 1b) suggest that phase transitions do not occur at this temperature within the probed pH range. The cooling curve shown in Figure 1c for KCl at pH 2 indicates a phase transition within a broad temperature range, while free-standing bilayers show a sharp main transition within ΔT smaller than 1 °C.48 The broadening is consistent with the coexistence of gel (Lβ) and fluid (Lα) phases in sBLMs,5,49 causing transitions at nonconstant tension.49 On the basis of this fact, Charrier and Thibaudau successfully modeled the broad transition of a DMPC bilayer on mica within a basic thermodynamic framework,49 while other authors suggest interpretations through finite-size-limited first-order transition models or interpret their data in the framework of a classic van’t Hoff transition.50 The lower transition temperatures observed for the DOPC membrane in KClO4 and MgCl2 solutions correspond to the increased membrane pressure due to the interaction of the ClO4− and Mg2+ ions with the lipid headgroup region (see Discussion above). Furthermore, the smaller transition range and the higher end points of the cooling curves suggest a different structure of the bilayer in both electrolytes. Considering again the coexistence of different phases in the sBLMs, the latter could result from the existence of different (gel) phases at pH 2 in KClO4 and MgCl2 solutions.

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ASSOCIATED CONTENT

S Supporting Information *

Diffusion coefficients of the DOPC membranes in dependence of the solution pH at varying anionic and cationic composition of the background electrolyte at an ionic strength of 1 mM, and additionally for CaCl2 and MgCl2 solutions at ionic strength of 10 and 100 mM, as well as zeta potential vs solution pH for different electrolytes of varied anionic/cationic composition at ionic strength of 0.1, 1, and 10 mM. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-351-4658-258. Fax: +49-351-4658-533. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Singer, S. J.; Nicolson, G. L. Science 1972, 175, 720−731. (2) Sackmann, E. In Structure and Dynamics of Membranes: From Cells to Vesicles; Lipowski, R.; Sackmann, E., Eds.; Elsevier: Amsterdam, The Netherlands, 1995; pp 213−304. (3) Felber, A. E.; Dufresne, M.-H.; Leroux, J.-C. Adv. Drug Delivery Rev. 2011, DOI: 10.1016/j.addr.2011.09.006. (4) Tenchov, B.; Koynova, R.; Rapp, G. Biophys. J. 2001, 80, 1873− 1890. (5) Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, T. Biophys. J. 2004, 86, 3783−3793. 6524

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(6) Träuble, H.; Eibl, H. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 214− 219. (7) Duman, J. G.; Lee, E.; Lee, G. Y.; Singh, G.; Forte, J. G. Biochemistry 2004, 43, 7924−7939. (8) Gurtovenko, A. A.; Vattulainen, I. J. Am. Chem. Soc. 2005, 127, 17570−17571. (9) McLaughlin, S. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113−136. (10) Böckmann, R. A.; Hac, A.; Heimburg, T.; Grubmüller, H. Biophys. J. 2003, 85, 1647−1655. (11) Vacha, R.; Siu, S. W. I.; Petrov, M.; Böckmann, R. A.; BaruchaKraszewska, J.; Jurkiewicz, P.; Hof, M.; Berkowitz, M. L.; Jungwirth, P. J. Phys. Chem. B 2010, 114, 9504−9509. (12) Chiantia, S.; Ries, J.; Kahya, N.; Schwille, P. ChemPhysChem 2006, 7, 2409−2418. (13) Zhou, Y.; Raphael, M. R. Biophys. J. 2007, 92, 2451−2462. (14) Gurtovenko, A. A.; Vattulainen, I. J. Phys. Chem. B 2008, 112, 4629−4634. (15) Jungwirth, P. Faraday Discuss. (Spiers Memorial Lecture) 2009, 141, 9−30. (16) Vlachy, N.; Jagoda-Cwiklik, B.; Vacha, R.; Touraud, D.; Jungwirth, P.; Kunz, W. Adv. Colloid Interface Sci. 2009, 146, 42−47. (17) Sachs, J. N.; Nanda, H.; Petrache, H. I.; Woolf, T. B. Biophys. J. 2004, 86, 3772−3782. (18) Sachs, J. N.; Woolf, T. B. J. Am. Chem. Soc. 2003, 125, 8742− 8743. (19) Garcia-Celma, J. J.; Hatahet, L.; Kunz, W.; Fendler, K. Langmuir 2007, 23, 10074−10080. (20) Zimmermann, R.; Küttner, D.; Renner, L.; Kaufmann, M.; Zitzmann, J.; Müller, M.; Werner, C. Biointerphases 2009, 4, 1−6. (21) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55−65. (22) Liu, Y.; Nagle, J. F. Phys. Rev. E 2004, 69, 040901−1−040901− 4. (23) Siu, S. W. I.; Vácha, R.; Jungwirth, P.; Böckmann, R. A. J. Chem. Phys. 2008, 128, 125103−1-125103−12. (24) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055−1069. (25) Soumpasis, D. M. Biophys. J. 1983, 41, 95−97. (26) Werner, C.; Kö rber, H.; Zimmermann, R.; Dukhin, S.; Jacobasch, H.-J. J. Colloid Interface Sci. 1998, 208, 329−346. (27) Zimmermann, R.; Osaki, T.; Schweiss, R.; Werner, C. Microfluid. Nanofluid. 2006, 2, 367−379. (28) Lyklema, J. Fundamentals of Interface and Colloid Science; Academic Press: London, U.K., 1991; Vol. II. (29) Lyklema, J. Fundamentals of Interface and Colloid Science; Academic Press: London, U.K., 1991; Vol. I. (30) Beattie, J. K. Lab Chip 2006, 6, 1409−1411. (31) Zangi, R.; Enberts, J. B. F. N. J. Am. Chem. Soc. 2005, 127, 2272−2276. (32) Zimmermann, R.; Freudenberg, U.; Schweiß, R.; Küttner, D.; Werner, C. Curr. Opin. Colloid Interface Sci. 2010, 15, 196−202. (33) Clarke, R. J.; Lüpfert, C. Biophys. J. 1999, 76, 2614−2624. (34) Scherer, P. G.; Seelig, J. Biochemistry 1989, 28, 7720−7728. (35) Böckmann, R. A.; Grubmüller, H. Angew. Chem., Int. Ed. 2004, 43, 1021−1021. (36) Cordomi, A.; Edholm, O.; Perez, J. J. J. Phys. Chem. B 2008, 112, 1397−1408. (37) Vernier, P. T.; Ziegler, M. J.; Dimova, R. Langmuir 2009, 25, 1020−1027. (38) Gutknecht, J. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 6443−6446. (39) Aroti, A.; Leontidis, E.; Dubois, M.; Zemb, T. Biophys. J. 2007, 93, 1580−1590. (40) Shirai, O.; Yoshida, Y.; Kihara, S. Anal. Bioanal. Chem. 2006, 386, 494−505. (41) Tatulian, S. A.; Gordeliy, V. I.; Sokolova, A. E.; Syrykh, A. G. Biochim. Biophys. Acta 1991, 1070, 143−151. (42) Pabst, G.; Hodzic, A.; Strancar, J.; Danner, S.; Rappolt, M.; Laggner, P. Biophys. J. 2007, 99, 2688−2696.

(43) Dörfler, H.-D.; Miethe, P.; Meyer, H. W. Chem. Phys. Lipids 1990, 54, 181−192. (44) Hauser, H. Chem. Phys. Lipids 1991, 57, 309−325. (45) Sinn, C. G.; Antonietti, M.; Dimova, R. Colloids Surf. A 2006, 282−283, 410−419. (46) Tatulian, S. A. Eur. J. Biochem. 1987, 170, 413−420. (47) Binder, H; Zschörnig, O. Chem. Phys. Lipids 2002, 115, 39−61. (48) Heimburg, T. Biochim. Biophys. Acta 1998, 1415, 147−162. (49) Charrier, A.; Thibaudau, F. Biophys. J. 2005, 89, 1094−1101. (50) Feng Xie, A.; Yamada, R.; Gewirth, A. A.; Granick, S. Phys. Rev. Lett. 2002, 89, 2461031−2461034.

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