The effect of metal cations on the phase behavior and hydration characteristics of phospholipid membranes

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Abstract

To characterize the specificity of ion binding to phospholipids in terms of headgroup structure, hydration and lyotropic phase behavior we studied 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of relative humidity (RH) at 25 °C in the presence and absence of Li+, Na+, K+, Be2+, Mg2+, Ca2+, Sr2+, Ba2+, Zn2+ and Cu2+ ions by means of infrared (IR) spectroscopy. All divalent cations and Li+ shift the gel-to-liquid crystalline phase transition towards bigger RH values indicating stabilization of the gel state. The observed shift correlates in a linearly fashion with the electrostatic solvation free energy for most of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was interpreted in terms of the excess chemical potential of mixing of hydrated ions and lipids. Calcium, zinc and partially lithium, cause a positive deviation from the linear relationship. IR spectral analysis shows that the carbonyl groups become more accessible to the water in the presence of Mg2+, Ca2+, Sr2+ and Ba2+ probably because of their involvement into the hydration shell of the ions. In contrast, Be2+, Zn2+ and Cu2+ dehydrate the carbonyl groups at small and medium RH. The ability of the lipid to take up water is distinctly reduced in the presence of Zn2+ and, partially, of Cu2+ meaning that the headgroups have become less hydrophilic. The binding mode of Be2+ to lipid headgroups involves hydrolyzed water. Polarized IR spectra show that complex formation of the phosphate groups with divalent ions gives rise to conformational changes and immobilization of the headgroups. The results are discussed in terms of the lyotropic Hofmeister series and of fusogenic activity of the ionic species.

Introduction

There is considerable interest in the mode of binding of metal cations to biological membranes because it affects stability and structure of phospholipid bilayers and modulates the binding and insertion of proteins. Ions interact with charged phospholipids via Coulombic forces. The apparent association of metal cations with lipid membranes is distinctly more intense for anionic lipids than for neutral, zwitterionic ones because the net negative surface charge of membranes of acidic lipids increases cation concentration near the lipid–water interface according to the Gouy–Chapman theory of the electrical double layer (McLaughlin et al., 1981). The respective intrinsic association constant of selected ions was, however, shown to be virtually independent of the net surface charge after correction for differences in electric surface potential (Altenbach and Seelig, 1984, Huster et al., 2000). It reflects specific lipid–ion interactions such as orientation dependent charge–dipolar interactions with neutral residues. In addition, molecular orbital effects can make an important contribution to the energetics and coordination of complexes with transition metals (Binder et al., 2001, Christianson, 1991). Also water potentially participates as a ligand in coordination of metal cations with lipid headgroups.

Ions can deeply penetrate of into the polar region of the membrane, which is an interphase rather than an interface because the polar residues are distributed throughout a meshlike region (Cevc, 1991). ‘Chemical’ factors give rise to the binding of ions to individual sites such as the phosphodiester, and possibly also to the carbonyl groups of the lipids. Such headgroup-ion complexes are highly specific and typically involve ions, polar and/or charged moieties of the lipid and hydration water as well (Garidel and Blume, 1999, Garidel et al., 2000). Different, equally charged metal cations can exhibit very different capabilities in affecting membrane properties, such as their ability to induce aggregation and fusion (Arnold, 1995, Ohki and Arnold, 1990, Ohki and Duax, 1986) and their potency to alter the surface and/or dipole potential and the surface pressure acting within the polar interface (Ermakov et al., 2001, McLaughlin et al., 1978, McLaughlin et al., 1981, Papahadjopoulos, 1968). Moreover, transition temperatures between gel and liquid-crystalline phases, and domain formation in mixed lipid membranes are specifically modulated by ion binding to the lipid headgroups (Garidel and Blume, 1999, Huster et al., 2000, Seelig et al., 1987, Silvius and Gagne, 1984).

Specific ion–phospholipid complexes are probably involved in the physiological role of metal cations. Numerous binding sites can buffer the ion concentration in cells. For example, retinal membranes have been suggested to bind a considerable fraction of the intracellular Ca2+ ions (Schnetkamp, 1985). Most likely, the buffer capacity of membranes for ions is an important factor that determines the distribution of ions within living cells (Ichikawa, 1996). Also Beryllium induces a broad spectrum of membranotropic effects in model systems, and in vivo. Long known for its high toxicity, the inhaled beryllium (or BeO) dust causes immune-mediated lesions in lungs (chronic beryllium disease), associated with lymphocyte infiltration and aggregation of macrophages (Finch et al., 1998). The specific role of zinc was recently discussed (Binder et al., 2000, Binder et al., 2001).

The present work deals with the specific nature of ion–phospholipid interactions. To this aim, we studied bilayers of the neutral zwitterionic lipid 1,2-palmitoyl-oleoyl-phosphocholine (POPC) as a function of water activity in the presence of chlorides of a series of monovalent alkali metal cations (Li+, Na+, K+) and of divalent alkaline earth (Be2+, Mg2+, Ca2+, Sr2+, Ba2+) and transition metal (Cu2+, Zn2+) ions, which differ in size and electronic structure. POPC was used because the absence of net charge allows focusing on specific lipid–ion interactions.

We studied lipid–ion interactions as a function of water activity. The investigation of amphiphilic systems at reduced hydration is a well suited technique for this issue because the distribution of ions between the membrane and water strongly shifts towards the lipid phase, and thus net effects of the added ions are intensified compared with highly diluted solutions. Moreover, the interaction of ions with solid and fluid membranes can be judged by their effect on the lyotropic gel-to-liquid crystalline phase transition of the lipid. Finally, studies at variable hydration allow assessing the role of water for ion–lipid interactions and the stability of amphiphilic systems. Relatively strong interactions of divalent cations such as Zn2+ with proteins and lipids are paralleled by considerable desolvation effects (Binder et al., 2001, DiTusa et al., 2001). It was recently shown that the enthalpy of ion–protein interactions mirrors the enthalpy of hydration (DiTusa et al., 2001). Consequently, hydration effects must be considered to interpret the association of ions to macromolecules, lipid membranes and other molecular aggregates in aqueous solution. In this study we combined hydration studies with polarized beam infrared (IR) attenuated total reflection (ATR) measurements, which are capable to characterize the molecular architecture of the polar interface, details of lipid hydration and specific lipid–ion interaction sites (Binder et al., 1997, Binder et al., 1998, Binder et al., 1999, Binder et al., 2001).

The paper is organized as follows, first, we examine the effect of the ions on the lyotropic chain melting transition of POPC. Second, binding of the ions to the phosphate and carbonyl groups and their conformational response were analyzed. Third, we characterize the effect of the ions on the hydration shell of the lipid. In the discussion section the results were correlated to the size and electronic structure of the ions. The results were also discussed in terms of Hofmeister series and fusogenic activity.

Section snippets

Materials and preparation of lipid vesicles

Vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti Polar Lipids, Alabaster, USA) were prepared as follows. The lipid in a chloroform/methanol stock solution (1:3 v/v), was dried and re-suspended by vortexing in water at final lipid concentration of 2–3 mg ml−1, followed by extrusion (Lipex extruder, Biomembranes, Vancouver, Canada) of the suspension through a polycarbonate Unipore membrane (100 nm pore-size, Millipore).

Sample solutions were prepared by mixing appropriate

The effect of metal ions on the lyotropic chain melting transition of POPC

The chain melting phase transition of phospholipids can be induced in an alternative fashion by increasing temperature, external pressure or hydration (Cevc and Marsh, 1987). We studied the lyotropic phase behavior of the lipid POPC in the absence and presence of various metal chlorides because it gets insight into details of interaction between the components water, lipid and metal chlorides (Binder et al., 1999b). Fig. 1 shows the COG of the symmetric methylene stretching band of the lipid

Lipid–ion complexes

Previous investigations on interactions of divalent cations such as Ca2+ and Zn2+ with phospholipid membranes indicated the formation of a well-defined chemical complex (Altenbach and Seelig, 1984, Binder et al., 2001, Huster et al., 2000). Its stochiometry depends on the type of metal cations, the lipid, its phase state (liquid-crystalline or gel) and on the water activity. The stochiometry refers to the molar ratio ion-to-lipid at saturation, RM/L(sat) (see Appendix A). For example, one Ca2+

Summary and conclusion

Insertion of most of the ions into the polar region of lipid membranes of zwitterionic POPC cause the shift of the lyotropic chain melting transition towards higher water activities. Consequently, ion–lipid interactions stabilize the solid phase of the lipid. The effect of the ions on the phase transition POPC correlates in a linearly fashion with the electrostatic solvation free energy of the ions in water that in turn, is inversely related to the ionic radius. This interesting result was

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