Swelling of phospholipids by monovalent salt

Abstract

Critical to biological processes such as membrane fusion and secretion, ion-lipid interactions at the membrane-water interface still raise many unanswered questions. Using reconstituted phosphatidylcholine membranes, we confirm here that multilamellar vesicles swell in salt solutions, a direct indication that salt modifies the interactions between neighboring membranes. By varying sample histories, and by comparing with data from ion carrier-containing bilayers, we eliminate the possibility that swelling is an equilibration artifact. Although both attractive and repulsive forces could be modified by salt, we show experimentally that swelling is driven primarily by weakening of the van der Waals attraction. To isolate the effect of salt on van der Waals interactions, we focus on high salt concentrations at which any possible electrostatic interactions are screened. By analysis of X-ray diffraction data, we show that salt does not alter membrane structure or bending rigidity, eliminating the possibility that repulsive fluctuation forces change with salt. By measuring changes in interbilayer separation with applied osmotic stress, we have determined, using the standard paradigm for bilayer interactions, that 1 M concentrations of KBr or KCl decrease the van der Waals strength by 50%. By weakening van der Waals attractions, salt increases energy barriers to membrane contact, possibly affecting cellular communication and biological signaling. Critical to biological processes such as membrane fusion and secretion, ion-lipid interactions at the membrane-water interface still raise many unanswered questions. Using reconstituted phosphatidylcholine membranes, we confirm here that multilamellar vesicles swell in salt solutions, a direct indication that salt modifies the interactions between neighboring membranes. By varying sample histories, and by comparing with data from ion carrier-containing bilayers, we eliminate the possibility that swelling is an equilibration artifact. Although both attractive and repulsive forces could be modified by salt, we show experimentally that swelling is driven primarily by weakening of the van der Waals attraction. To isolate the effect of salt on van der Waals interactions, we focus on high salt concentrations at which any possible electrostatic interactions are screened. By analysis of X-ray diffraction data, we show that salt does not alter membrane structure or bending rigidity, eliminating the possibility that repulsive fluctuation forces change with salt. By measuring changes in interbilayer separation with applied osmotic stress, we have determined, using the standard paradigm for bilayer interactions, that 1 M concentrations of KBr or KCl decrease the van der Waals strength by 50%. By weakening van der Waals attractions, salt increases energy barriers to membrane contact, possibly affecting cellular communication and biological signaling. From bacteria to mammals, biological processes take place in salt solutions. Maintaining a delicate balance of ions, biomembranes interact preferentially with different ionic species (1Reynolds J.A. Are inorganic cations essential for stability of biological membranes?.Ann. N. Y. Acad. Sci. 1972; 195: 75-85Google Scholar). Specific ionic effects have been shown to influence the growth rates of bacteria (2Lo Nostro P. Ninham B.W. Lo Nostro A. Pesavento G. Fratoni L. Baglioni P. Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa.Phys. Biol. 2005; 2: 1-7Google Scholar) and fungi (3Keppler F. Eiden R. Niedan V. Pracht J. Scholer H.F. Halocarbons produced by natural oxidation processes during degradation of organic matter.Nature. 2000; 403: 298-301Google Scholar, 4Poolman B. Spitzer J.J. Wood J.A. Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions.Biochim. Biophys. Acta. 2004; 1666: 88-104Google Scholar) and to affect the function of antibiotic channels (5Grigorjev P.A. Bezrukov S.M. Hofmeister effect in ion transport: reversible binding of halide anions to the roflamycoin channel.Biophys. J. 1994; 67: 2265-2271Google Scholar). This investigation will focus on the halide salts because of their presence in intracellular and intercellular fluids. At biomembrane surfaces, halides encounter not only charged lipid species, such as phosphatidylserine and phosphatidylinositol, but more often common neutral phosphatidylcholines (PCs). Interactions with both types of lipids require investigation: by affecting lipid interactions, salt solutions modulate biological processes such as fusion and secretion. Despite being electrically neutral, PC membranes attract one another as a result of mutually induced charge fluctuations (6Rand R.P. Parsegian V.A. Hydration forces between phospholipid bilayers.Biochim. Biophys. Acta. 1989; 988: 351-376Google Scholar). Because of different dielectric properties of membranes and the intervening solvent, transient spontaneous electromagnetic fields in one membrane induce correlated fields in the neighboring membrane and vice versa, resulting in an attractive force. This “charge fluctuation” (van der Waals) force is responsible for the spontaneous formation of stable multilamellar structures [e.g., myelin sheets in vivo and multilamellar vesicles (MLVs) in vitro]. When the attractive van der Waals force is exactly balanced by repulsive forces, an equilibrium spacing between lamellae is established (6Rand R.P. Parsegian V.A. Hydration forces between phospholipid bilayers.Biochim. Biophys. Acta. 1989; 988: 351-376Google Scholar, 7McDaniel R.V. McIntosh T.J. Simon S.A. Non-electrolyte substitution for water in phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1983; 731: 97-108Google Scholar, 8McIntosh T.J. Simon S.A. Contributions of hydration and steric (entropic) pressures to the interactions between phosphatidylcholine bilayers: experiments with the subgel phase.Biochemistry. 1993; 32: 8374-8384Google Scholar, 9Nagle J.F. Tristram-Nagle S. Structure of lipid bilayers.Biochim. Biophys. Acta. 2000; 1469: 159-195Google Scholar). Typically, as measured by small-angle X-ray scattering, the interlamellar spacing of neutral membranes in water is on the order of the membrane thickness itself. However, membrane spacings depend sensitively on the nature of the solvent (7McDaniel R.V. McIntosh T.J. Simon S.A. Non-electrolyte substitution for water in phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1983; 731: 97-108Google Scholar, 10Korreman S.S. Posselt D. Modification of anomalous swelling in multilamellar vesicles induced by alkali halide salts.Eur. Biophys. J. 2001; 30: 121-128Google Scholar, 11Deme B. Dubois M. Zemb T. Swelling of a lecithin lamellar phase induced by small carbohydrate solutes.Biophys. J. 2002; 82: 215-225Google Scholar). Therefore, any alteration of the repeat spacing with solvent composition is an indication of a shift in the balance of attractive and repulsive forces between membranes. Here, we specifically address the modification of interbilayer forces by monovalent salt. Modification of interbilayer forces by solvents and solutes, including monovalent salt, has been recognized in the past (7McDaniel R.V. McIntosh T.J. Simon S.A. Non-electrolyte substitution for water in phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1983; 731: 97-108Google Scholar, 10Korreman S.S. Posselt D. Modification of anomalous swelling in multilamellar vesicles induced by alkali halide salts.Eur. Biophys. J. 2001; 30: 121-128Google Scholar, 12Gottlieb M.H. Eanes E.D. Influence of electrolytes on thicknesses of phospholipid bilayers of lamellar lecithin mesophases.Biophys. J. 1972; 12: 1533-1539Google Scholar, 13Chapman D. Peel W.E. Kingston B. Lilley T.H. Lipid phase transitions in model biomembranes. The effect of ions on phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1977; 464: 260-275Google Scholar, 14Cunningham B.A. Shimotake J.E. Tamuralis W. Mastran T. Kwok W.M. Kauffman J.W. Lis L.J. The influence of ion species on phosphatidylcholine bilayer structure and packing.Chem. Phys. Lipids. 1986; 39: 135-143Google Scholar, 15Cunningham B.A. Lis L.J. Interactive forces between phosphatidylcholine bilayers in monovalent salt solutions.J. Colloid Interface Sci. 1989; 128: 15-25Google Scholar, 16Simon S.A. McIntosh T.J. Magnitude of the solvation pressure depends on dipole potential.Proc. Natl. Acad. Sci. USA. 1989; 86: 9263-9267Google Scholar). However, outstanding questions remain: What is the swelling mechanism? What is the nature of ionic specificity? Does salt affect membrane structure in addition to interactions? With recent advances in X-ray methods for the determination of bilayer structure and interactions (17Tristram-Nagle S. Nagle J.F. Lipid bilayers: thermodynamics, structure, fluctuations, and interactions.Chem. Phys. Lipids. 2004; 127: 3-14Google Scholar, 18Liu Y. Nagle J.F. Diffuse scattering provides material parameters and electron density profiles of biomembranes.Phys. Rev. E. 2004; 69: 40901Google Scholar, 19Kučerka N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar), we are now in a position to address salt effects on multilamellar lipid structures. Significant ion-lipid interactions are expected not only because of dielectric gradients across the biomembrane interface but also because of the dipolar nature of head groups. Ion-lipid interactions have been measured by solid-state NMR spectroscopy (20Brown M.F. Seelig J. Ion-induced changes in head group conformation of lecithin bilayers.Nature. 1977; 269: 721-723Google Scholar), electrophoretic mobility (21Eisenberg M. Gresalfi T. Riccio T. McLaughlin S. Adsorption of monovalent cations to bilayer membranes containing negative phospholipids.Biochemistry. 1979; 18: 5213-5223Google Scholar, 22Tatulian S.A. Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes.Eur. J. Biochem. 1987; 170: 413-420Google Scholar, 23Cohen J.A. Electrophoretic characterization of liposomes.Methods Enzymol. 1995; 367: 148-176Google Scholar), and monolayer surface pressure (24Aroti A. Leontidis E. Maltseva E. Brezesinski G. Effects of Hofmeister anions on DPPC Langmuir monolayers at the air-water interface.J. Phys. Chem. B. 2004; 108: 15238-15245Google Scholar). The electrostatic nature of ion-lipid interactions has raised experimental questions as well, highlighting possible equilibration complications, such as the case of Li salts (25Rappolt M. Pressl K. Pabst G. Laggner P. Lα-phase separation in phosphatidylcholine-water systems induced by alkali chlorides.Biochim. Biophys. Acta. 1998; 1372: 389-393Google Scholar) or high-temperature melting lipids (26Gruner S.M. Lenk R.P. Janoff A.S. Ostro M.J. Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles.Biochemistry. 1985; 24: 2833-2842Google Scholar). Here, we show that the swelling of PC bilayers by monovalent salt is driven mainly by the weakening of van der Waals attractive forces through screening of charge fluctuations. MLVs swell more in the presence of Br− compared with Cl− as a result of a higher propensity of bromine ions to associate with the polar lipid head groups (22Tatulian S.A. Binding of alkaline-earth metal cations and some anions to phosphatidylcholine liposomes.Eur. J. Biochem. 1987; 170: 413-420Google Scholar, 27Tatulian S.A. Effect of lipid phase on the binding of anions to Biophys. Acta. 1983; Scholar, S.A. diffraction of the influence of ions on phospholipid membrane interactions.Biochim. Biophys. Acta. Scholar, M. McLaughlin S. of the electrostatic to model phospholipid J. 1995; Scholar, of binding to neutral lipid membranes using Scholar, Influence of anions and cations on the dipole of phosphatidylcholine vesicles: a for the Hofmeister J. Scholar), an electrostatic between the now charged To isolate the effect of salt on van der Waals interactions, we focus on interbilayer forces in the presence of high salt concentrations at which the electrostatic be that the bilayer structure and bending are by salt, we to osmotic pressure data at high salt with that ions biomembrane interactions not only through electrostatic interactions but also by a screening of charge fluctuations. By biomembrane ionic at the biomembrane interface a for and and from and solvents from KCl and KBr salts of from lipids hydrated with water or salt solutions. When osmotic pressure the high or of and the melting or and for at in a with or or to the of sample on being to X-ray for with a X-ray to between 1 and in the of the or scattering position scattering of sample equilibration at spacings as a function of applied osmotic as by N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar). The that salt to the organic solvent from which the oriented using the S. R. Nagle J.F. of in fully hydrated bilayers of phase J. 1993; Scholar). The of salt that that the of salt in the interlamellar water at hydration that of the X-ray data for oriented at the of the using the sample and the by N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar). for and the the scattering is by the standard Nagle J.F. of the structure of lipid Rev. E. 1994; 1 structure and to The bilayer is the of the electron density across a bilayer in water with the fluid electron density for and applied to Y. Nagle J.F. Diffuse scattering provides material parameters and electron density profiles of biomembranes.Phys. Rev. E. 2004; 69: 40901Google Scholar, Y. Liu Y.F. Tristram-Nagle S. J. Nagle J.F. for structure and interactions from oriented lipid Rev. E. 2001; Scholar), DLPC N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar), and DMPC N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar). The is to the of the scattering in the to the bending and the are the material parameters that the fluctuations and the of The is for a by the fluctuations J.F. Tristram-Nagle S. Structure of lipid bilayers.Biochim. Biophys. Acta. 2000; 1469: 159-195Google Scholar) and to the model of the electron density Nagle J.F. Structure of the fully hydrated phase of J. 1989; Scholar). is from the J.F. Structure of fully hydrated bilayer Biophys. Acta. Scholar, J.F. for lipid of electron density profiles to J. 1989; the of is for the electron density of water is and the of the lipid is for DLPC using the neutral J.F. and J. Scholar, Tristram-Nagle S. Nagle J.F. Specific of lipids in fully hydrated bilayer Biophys. Acta. Scholar). the case of DLPC with salt, the of the interlamellar solvent and the at A. E. properties of and solutions of and at from to at of and Chem. Scholar, der in Scholar). The electron of salt water for DLPC with M interlamellar KCl 1 M and for DLPC with M interlamellar KBr M The bilayer and are some parameters that are from the model N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar). repeat spacings for the DLPC in KCl and KBr solutions at are shown in 1 as of salt To the change of scattering with salt, we also show X-ray in high salt of position data to salt concentrations of M for KCl and M for From an equilibrium of in DLPC swell with salt, an of for the for KCl is The DLPC lipid is not a PC lipids with different also swell with monovalent salt, as shown in the measured repeat spacing only the of interlamellar water spacing by the bilayer thicknesses J.F. Tristram-Nagle S. Structure of lipid bilayers.Biochim. Biophys. Acta. 2000; 1469: 159-195Google Scholar, 19Kučerka N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar, H.I. M.F. lipid and in fluid phosphatidylcholines by NMR J. 2000; Scholar). Although a for the membrane thickness is for the analysis of membrane interactions for comparison with the the more common that with the of lipid and lipid to an in water spacings with of or with the of spacing and water thickness M KCl or KBr to the fluid of DLPC and By membrane thicknesses from the comparison of the resulting water spacings in that swelling with salt is by the from to or by a is that multilamellar structures not fully in salt solutions because lipid membranes have to ions, to the water in of unilamellar vesicles often liposomes are of vesicles affecting in phospholipid vesicles by the a possible for the of Phys. Lipids. Scholar). gradients as a result of and However, in is that salt does the water of salt an osmotic pressure that to the water spacing in with the with membranes as is that as measured by X-ray is stability at for to X-ray for at of the for the sample to at the is also equilibration for oriented as This that ions through of multilamellar equilibration in the presence of the common and changes in interlamellar spacings or equilibration for of by at higher but are to alteration of membrane structure and interactions to changes in equilibration the that are not in energy is measured sample with and the To salt membrane structure and we have on oriented DLPC the scattering from an oriented sample of DLPC to The of the are in for M and M KBr at salt concentrations to have the lamellar as in are small but the for the which that the bilayer structure is different in different salt solutions. However, the electron density profiles in that from show that the bilayer structures are The in the are to in the electron density of the interlamellar water resulting from the addition of salt. This changes the electron density between lipids and salt solutions the electron density of the of are data and are model to the fluctuations the are to density profiles of M and M KBr at of the bilayer is the in electron of the different solvents that for of the in the in To the and the electron density we the membrane bending and the which is a of hydration forces, van der Waals forces, and the fluctuation force. The are in as a function of for at not with salt or The are in the does as a function of N. N. Liu Y.F. Tristram-Nagle S. Nagle J.F. swelling of lipid bilayer is by of the bending Rev. E. 2005; Scholar, H.I. N. Tristram-Nagle S. Nagle J.F. interactions from E. 1998; Scholar). are in from are with a as of parameters for bilayer that are in 1 to the in M M in a The of interactions between bilayers is by osmotic pressure and measuring water spacing a to data (6Rand R.P. Parsegian V.A. Hydration forces between phospholipid bilayers.Biochim. Biophys. Acta. 1989; 988: 351-376Google Scholar). Here, we have to a as the steric water spacing a is the steric thickness of the bilayer of the At osmotic at from the DLPC structure determination N. Liu Y.F. Chu N.J. Petrache H.I. Tristram-Nagle S. Nagle J.F. Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles.Biophys. J. 2005; 88: 2626-2637Google Scholar). high osmotic the standard to membrane thickness (6Rand R.P. Parsegian V.A. Hydration forces between phospholipid bilayers.Biochim. Biophys. Acta. 1989; 988: 351-376Google Scholar) applied using an W. McIntosh T. D. E. Effect of and on of lipid J. 2000; Scholar). data for MLVs of DLPC with salt and with 1 M KCl and 1 M The of interbilayer interactions for fluid phase membranes a van der Waals attractive force and repulsive hydration and bending fluctuations. Although the attractive van der Waals force be for a of of thickness V.A. Ninham B.W. of to the of van der Waals forces across lipid Scholar), the repulsive forces are through parameters and of interbilayer interactions H.I. N. Tristram-Nagle S. Nagle J.F. interactions from E. 1998; The the van der Waals with an strength by the and we as The the hydration for the of water in the of lipid head groups. This force is with a (6Rand R.P. Parsegian V.A. Hydration forces between phospholipid bilayers.Biochim. Biophys. Acta. 1989; 988: 351-376Google Scholar, 16Simon S.A. McIntosh T.J. Magnitude of the solvation pressure depends on dipole potential.Proc. Natl. Acad. Sci. USA. 1989; 86: 9263-9267Google Scholar, H.I. N. Tristram-Nagle S. Nagle J.F. interactions from E. 1998; Scholar). the interbilayer force for small water spacings and for interbilayer spacings The in is the fluctuation or which the repulsive at water spacings T.J. Simon S.A. Contributions of hydration and steric (entropic) pressures to the interactions between phosphatidylcholine bilayers: experiments with the subgel phase.Biochemistry. 1993; 32: 8374-8384Google Scholar). for the to the of membranes W. of fluid membranes in Scholar). This force is to the bilayer bending rigidity, The and depend sensitively on the of membrane thickness as but that is be the strength of repulsive interactions that the effect of salt is at osmotic pressure but small at high osmotic at which the force between bilayers is the hydration force. Therefore, that salt the hydration force in a This is not a because the of salt to as the water spacing However, the hydration force is for in the in which salt is interactions be Therefore, we the of and to the high osmotic pressure data for solutions and to the shown in 1 in the The shown in the fluctuation forces in the we H.I. N. Tristram-Nagle S. Nagle J.F. interactions from E. 1998; Scholar) that the is by the fluctuation of the The experimental that is the and that the on the in that the parameters and for the repulsive fluctuation force be the for solutions. the to the data shown in provides the parameters and as shown in Therefore, the standard paradigm of the only that for the swelling of with salt is the of the attractive van der Waals 1 the of that by of the osmotic pressure data the parameters to the as The to the data are shown in in show that salt has effects on the thickness of DLPC Therefore, the for the swelling of multilamellar arrays with salt shown in is swelling of the water between the with the of bilayer structure, the measured bilayer in also does not change with salt. Because interbilayer forces from interactions, the model in has been as the for of experimental have experimentally interbilayer parameters by measuring the osmotic pressure the membrane bending and the interlamellar and not a experimental the of with interlamellar spacing is by salt, that fluctuation parameters are not the of is that a change of the is to the osmotic pressure data in 1 the of This that the is salt is to multilamellar This is a direct experimental of the V.A. Ninham B.W. of to the of van der Waals forces across lipid Scholar, V.A. van der Waals Scholar) by and Posselt S.S. Posselt D. Modification of anomalous swelling in multilamellar vesicles induced by alkali halide salts.Eur. Biophys. J. 2001; 30: 121-128Google Scholar) to their The strength of the van der Waals is to the dielectric between lipids and the the the the between bilayers V.A. Ninham B.W. of to the of van der Waals forces across lipid Scholar). To dielectric is modified by salt, we between and dielectric of V.A. parameters for of van der Waals Colloid Interface Sci. Scholar). are modified by a of ions, are modified by ionic in to spontaneous charge fluctuations. at is responsible for a decrease in the van der Waals for both KCl and The swelling in the presence of KBr compared with KCl at salt is to electrostatic from the binding of Br− ions H.I. D. Parsegian V.A. of ions at the biomembrane interface.J. Chem. 2005; 127: Scholar). This electrostatic in 1 M salt, at which the screening is With the energy of the multilamellar From of salt between bilayers is a salt in the the equilibrium spacing is established by the in the bilayer attraction. a fully in water a salt as in of salt the interlamellar the interlamellar water of salt in the presence of 1 M salt in the an osmotic salt to van der Waals is This the membranes to to a equilibrium at spacing To the spacing the energy to as from the pressure in of forces between bilayers, of salt is by the between salt and lipid head groups for interlamellar by neutral H.I. D. Parsegian V.A. of ions at the biomembrane interface.J. Chem. 2005; 127: Scholar), salt the MLVs is in the This is by a of water to the lipid head groups and to salt is from the of PC head with the Br− being At the cellular ionic effects have been recognized primarily for B. Spitzer J.J. Wood J.A. Bacterial osmosensing: roles of membrane structure and electrostatics in lipid-protein and protein-protein interactions.Biochim. Biophys. Acta. 2004; 1666: 88-104Google Scholar) and growth rates of bacteria (2Lo Nostro P. Ninham B.W. Lo Nostro A. Pesavento G. Fratoni L. Baglioni P. Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa.Phys. Biol. 2005; 2: 1-7Google Scholar). Here, we that the effect of salt on lipid will such cellular membrane The and for many on interbilayer This by the of the of of and for the by of data from oriented X-ray at the is