Specificity and Competitive Cation Association to Phosphatidylinositol-4,5-Bisphosphate Model Membranes

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Phosphatidylinositol-4,5-bisphosphate (PIP2) is an active signaling lipid implicated, among other functions, in the regulation of cell growth by activating the tumor suppressor PTEN. Using synchrotron surface-sensitive x-ray diffraction and fluorescence techniques we determined the preferential cation binding to PIP2 monolayers. The natural, highly unsaturated PIP2 was spread as a Langmuir monolayer on a physiological buffer containing 100 mM KCl at pH 7.2 to which divalent cations (Ca2+ and Mg2+) were added. X-ray fluorescence of the PIP2 monolayer on the buffer shows an eight fold surface enhancement (within the x-ray penetration depth below the critical angle, ∼5 nm) of monovalent K+ compared to its bulk concentration. When physiological levels of calcium are added (1-100 µM), the Ca2+ gradually replaces bound K+ ions, leading to a significant change in the organization of the PIP2 model membrane. At higher concentrations (100-1000 µM), which might be achieved during calcium signaling, we observe a 1000 fold surface enhancement of Ca2+. Similar experiments with Mg2+ ions also show strong ion binding to PIP2 at physiological levels (1 mM) with a lesser structural effect on the monolayer compared to that induced by Ca2+. For mixed solutions of Mg2+ and Ca2+ we find that Ca2+ occupies the majority of binding sites, and at mM concentrations completely removes the Mg2+ ions from the interface. Surprisingly, with both 1 mM Mg2+ and 1 mM Ca2+ in the subphase there is still a fourfold surface enrichment of K+ ions at the headgroup region.