Competitive cation binding to phosphatidylinositol-4,5-bisphosphate domains revealed by X-ray fluorescence

Abstract

Phosphatidylinositol-4,5-bisphosphate (PIP₂) is an important signaling phospholipid in the inner leaflet of the cell membrane. Due to the high negative charge of its headgroup, PIP₂ strongly interacts with cellular cations. We have used synchrotron diffraction and fluorescence techniques to determine preferential cation binding to two-dimensional PIP₂ templates. The natural, highly unsaturated PIP₂ is manipulated as a Langmuir monolayer on a physiological buffer containing 100 mM KCl and varying amounts of Ca²⁺ and Mg²⁺. X-ray fluorescence shows an 800% surface enhancement in K⁺ concentration (bound to PIP₂) compared to bulk concentration. Adding physiological levels of Ca²⁺( 1–100 μM) results in gradual replacement of K⁺ by Ca²⁺ ions, leading to a significant change in the organization of the PIP₂ model membrane, while higher concentrations (100–1000 μM) lead to three orders of magnitude increase in surface [Ca²⁺]. Similar experiments with Mg²⁺ ions also show strong ion binding to PIP₂ at physiological levels (1 mM) with a lesser structural effect. For mixed solutions of Mg²⁺ and Ca²⁺ we find that Ca²⁺ occupies the majority of binding sites. Remarkably we find that, with both 1 mM Mg²⁺ and 1 mM Ca²⁺ in the subphase there is still a 400% surface enrichment of K⁺ ions at the headgroup region.