Discussion

4.3.1. Chaotropic Anions are attracted to the Lipid Interface.

If anions are classified according to their free energy of hydration ΔGhyd (Marcus, 1991) a well defined series for the translocated charge is obtained where the anion with the smallest -ΔGhyd yields the largest negative charge translocation. This means the most hydrophobic or the most chaotropic anion binds best to or at the lipid.

These results exactly fit the series obtained for the influence of anions on the dipole potential of PC liposomes (Clarke & Lupfert, 1999). Clarke and Lüpfert explain the reduction of the dipole potential by binding of the chaotropic anion at the positive side of the dipole deep in the lipid headgroup. This is in agreement with our results, which show a large negative charge translocation upon binding of chaotropic anions like ClO₄^-.

4.3.2. Kosmotropic Cations are attracted to the Lipid Interface.

Interestingly, for cations the inverse series is obtained with respect to -ΔGhyd. We found that the higher -ΔGhyd is, the larger the translocated charge. This means the most hydrophilic or the most kosmotropic cation binds best. Again, our results agree with those obtained for the effect of cations on the lipid dipole potential (Clarke &

Lupfert, 1999) but also with an early study of the adsorption of cations to phosphatidylserine liposomes (Eisenberg, 1979). In consequence, we find a more attractive potential for kosmotropic than for chaotropic cations.

4.3.3. PC Membranes can be Anion or Cation Selective.

Up to now we have only ranked ion binding within the group of anions or cations. The polarity of the charge displacements after uncompensated salt concentration jumps, however, indicates whether cations or anions bind preferentially to the membrane.

These experiments show that a zwitterionic PC surface (no net charge) depending on the specific ion pair can prefer the cation or the anion. In particular, the strongly chaotropic anions (SCN^- and ClO₄^-) have a higher affinity for the membrane than their Na⁺ counter ions, while for the chlorides NaCl and KCl the cations bind stronger. This is unexpected because Hofmeister interactions are generally believed to be dominated by anions (Collins & Washabaugh, 1985). Finally, the uncompensated salt concentration jump experiments also rule out that the specific cation effects (Fig. 7)

SPECIFIC ANION AND CATION BINDING TO LIPID MEMBRANES

are brought about predominantly by extraction of tightly bound Cl^- from the surface (yielding a positive charge displacement), rather than binding of the cations.

4.3.4. Headgroup Charges make a Difference.

The most abundant lipid in an average mammalian cell membrane is phosphatidylcholine (45 – 55%), which comprises a negatively charged phosphate (PO4-) group and a positively charged ammonium (N(CH3)3+) group. But also the charged sites of many lipid membrane constituents are phosphate and ammonium groups. Consequently, PC is a good prototype lipid for a study of the interaction of ions with a biological membrane. To address the question how these groups determine the interaction of ions with the SSM surface, a membrane composed of the zwitterionic PC is compared to a membrane composed of a lipid with no charged groups (Mono). Additionally, net charged lipids, like the positively charged DOTAP or the negatively charged PG, were investigated.

From the experiments using lipids with different headgroups a clear tendency emerges that cations preferentially bind to the negatively charged PG while anions prefer the positively charged DOTAP headgroup. This is by itself not surprising and the standard Gouy-Chapman electrostatic theory describes accumulation of positive ions at negatively charged surfaces and vice versa. But we have to bear in mind that the observed charge translocations represent the difference to the reference ions Na⁺ and Cl^-. In the Gouy-Chapman formalism all ions of identical valency yield the same result and there should be no difference between ClO4- and Cl^- or between Na⁺ and K⁺. However, the effect of charged headgroups could be explained by the increased local cation concentration at the DOPG surface and the increased local anion concentration at the DOTAP surface predicted by the electrostatic interaction. It cannot, however, explain the more efficient binding to zwitterionic PC as compared to uncharged Mono, which is more prominent for the anions than for the cations (Fig. 7).

Taken together this seems to be suggestive of a mechanism different from pure electrostatics which modifies ion binding at different lipid headgroups.

4.3.5. Interaction Mechanism of Anions and Cations with a Lipid Interface.

Different physicochemical parameters of the ions like the Gibbs free energy of hydration chosen in this publication have been taken into account to rationalize the ionic sequences. Unfortunately many of these parameters are correlated themselves

SPECIFIC ANION AND CATION BINDING TO LIPID MEMBRANES

(Kunz et al., 2004) so that these procedures are of little help to identify the dominating forces acting between the interface and the ion. It has recently been shown that dispersion forces have to be taken into account at physiological ion concentrations where electrostatic interactions are screened (Kunz et al., 2004) and may be responsible for Hofmeister effects (Bostrom et al., 2002). The relevant parameter determining dispersion forces on ions is the ionic polarizability, which is large for large chaotropic ions (Kunz et al., 2004; Leontidis, 2002) and would predict preferential binding of both, chaotropic cations and anions, in disagreement with our experimental results.

An ion binding mechanism according to the ‘principle of matching water affinities’ has been proposed by Collins (Collins, 2004; Collins, 2006) which predicts binding of chaotropic ions to chaotropic surface groups and kosmotropic ions to kosmotropic surface groups. Since PO₄^- is strongly kosmotropic (and the putative binding site for cations) and N(CH₃)₃⁺ is chaotropic (and the putative binding site for anions) this would explain our results on PC. But it cannot account for the fact that a lipid without these residues (Mono) shows the same cation and anion binding series. And it cannot explain either why for various phosphate compounds as well as other anionic surfactants the reversed cation series is found (see. e.g. (Haverd & Warr, 2000) and the literature cited herein). In contrast to these compounds, however, in the present study the phosphate group is chemically linked to ammonium or glycerol groups or consists only of a glycerol headgroup (Mono), and, therefore, the influence of the phosphate group alone is not decisive. We believe, in agreement with the results of Jungwirth and co-workers (Jungwirth & Tobias, 2001; Vrbka et al., 2004) that it is necessary to precisely describe the role of the component that is present in larger excess, namely water. To understand the specific ion effects, it is very probably not sufficient to describe the water as a bulk or as hydration water. The geometry of the water molecules around the ions and headgroups very probably makes the difference. Advances in this direction come from recent molecular dynamics studies of zwitterionic PC membranes (Bockmann et al., 2003; Pandit et al., 2003; Sachs et al., 2004; Sachs & Woolf, 2003) and negatively (Mukhopadhyay et al., 2004; Zhao et al., 2007) or positively (Gurtovenko et al., 2005) charged lipid membranes. For example, simulation of PC membranes show that Cl^- binds slightly weaker than Na⁺ (Bockmann et al., 2003; Pandit et al., 2003) and that chaotropic anions bind better than kosmotropic ones to the lipid headgroups (Sachs et al., 2004; Sachs & Woolf, 2003). Both results agree with our experiments.

RAPID ACTIVATION OF MELB