Phosphatidylinositol-4,5-bisphosphate ionization in the presence of cholesterol, calcium or magnesium ions

•Ca2+ has a much larger effect on the ionization state of PI(4,5)P2 than Mg2+ at the same concentration.•Cholesterol effects the ionization of PI(4,5)P2 via a direct interaction with the headgroup of PI(4,5)P2.•Interactions of cholesterol, Ca2+ and Mg2+ affect the ionization of the 5-phosphate more than that of the 4-phosphate. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is an important signaling lipid and plays a crucial role in a wide variety of cellular processes by interacting with protein targets and localizing proteins at the plasma membrane. These interactions are strongly influenced by the lateral distribution of PI(4,5)P2 as well as its ionization state. The characterization of the PI(4,5)P2 ionization state provides important information about how PI(4,5)P2 interacts with other membrane resident or associated chemical species. In this study we have used solid-state MAS 31P NMR to investigate the interactions of PI(4,5)P2 with potential cluster promoting agents, divalent cations and cholesterol. Both Ca2+ and cholesterol were found previously to promote formation of local PI(4,5)P2 clusters in vitro. The NMR approach allows us to probe independently the ionization state of PI(4,5)P2 two phosphomonoester groups. We investigated mixed phosphatidylcholine (PC)/PI(4,5)P2 multilamellar vesicles in the presence of micro and millimolar concentrations of Ca2+ and Mg2+. We found that both cations lead to an increased downfield chemical shift of the PI(4,5)P2 phosphomonoester peaks, indicating an increased ionization in the presence of the divalent cations. Ca2+ has a much larger effect on PI(4,5)P2 as compared to Mg2+ at similar concentrations. Physiological concentrations of Ca2+ are significantly lower than those found for Mg2+ and the comparison of the PI(4,5)P2 ionization in the presence of Ca2+ and Mg2+ at physiological concentrations resulted in similar charges of the phosphomonoester groups for both cations. PI(4,5)P2 was also examined with vesicles containing cholesterol since cholesterol has been shown to promote PI(4,5)P2 clustering. In the presence of 40mol% cholesterol, the PI(4,5)P2 phosphomonoester 31P NMR peaks shifted slightly downfield, indicating a small increase in charge. Previously published data suggest that PI(4,5)P2 is capable of forming an intra- and intermolecular hydrogen bond network, which leads to a reduction of the charge at the phosphomonoester groups through dissipation of the charge across the bilayer/water interface