The Competitive Binding of Ca 2+ vs. Mg 2+ to PIP2-Containing Lipid Monolayers and the Comparison of Pi(4,5)P2 and its Isomers

Abstract

Interactions between divalent cations and PtdIns(4,5)2-containing lipid model membrane are investigated. Surface pressure measurements of lipid monolayers formed by binary lipid mixtures containing 25mol% PIP2 were used to quantify divalent cation binding. Direct titration and competitive binding assays show that divalent cations including Mg2+, Ca2+, Sr2+, Ba2+, Co2+, Ni2+, Cu2+, Zn2+ and some multivalent polyamines bind PIP2 with Kd ranges from 200nM-50uM. Some of these cations, which are considered physiologically important, were examined more closely and evidence for divalent cation-induced lateral segregation of PIP2 is presented. Such studies are based on fluorescence and atomic force microscopic studies of Langmuir-Schaeffer lipid monolayers, and are coupled with numerical studies. Ca2+ and Mg2+ have similar binding affinity to PIP2-containing monolayers, but Ca2+ induces PIP2 lateral segregation more efficiently than compared to Mg2+. Ca2+ vs. Mg2+ competitive binding assays are also carried out on PtdIns(3,4)P2- and PtdIns(3,5)P2-containing monolayers. Significant differences in the response of these three PIP2 isomers to divalent cations are found. Besides monolayer studies, PIP2-cation interactions are also examined on a bilayer system. Fluorescence correlation spectroscopy (FCS) and laser scanning confocal microscopy (LSCM) are used following divalent cation titration on asymmetric labeled PIP2-containing giant unilamellar vesicles (GUVs). The diffusion coefficient and the fluorescence intensity variance of fluorescent PIP2 change with increasing divalent cation concentration. Förster resonance energy transfer (FRET) studies using bi-color labeled PIP2 are also carried out in a large unilamellar vesicle (LUV) system. These results together reveal that the lateral inhomogeneity of PIP2 changes with divalent cation concentration on bilayer model membranes. These results may provide insight into divalent cation-induced PIP2 microdomain formation in the cell membrane.