title: Phosphatidylinositol-4,5-bisphosphate ionization in the presence of cholesterol, calcium or magnesium ions.
abstract: 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 (31)P NMR to investigate the interactions of PI(4,5)P2 with potential cluster promoting agents, divalent cations and cholesterol. Both Ca(2+) 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 Ca(2+) and Mg(2+). 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. Ca(2+) has a much larger effect on PI(4,5)P2 as compared to Mg(2+) at similar concentrations. Physiological concentrations of Ca(2+) are significantly lower than those found for Mg(2+) and the comparison of the PI(4,5)P2 ionization in the presence of Ca(2+) and Mg(2+) 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 40 mol% cholesterol, the PI(4,5)P2 phosphomonoester (31)P 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. We hypothesize that cholesterol participates in this intermolecular hydrogen bond network, resulting in a stabilization of PI(4,5)P2 enriched domains due an increased spacing between the PI(4,5)P2 headgroup. We also examined the cumulative effects of cholesterol combined with the divalent cations, phosphatidylethanolamine (PE), and phosphatidylinositol (PI), separately. The combination of cholesterol and divalent cations results in an additive effect on PI(4,5)P2 ionization, while the effect of cholesterol on PI(4,5)P2 ionization is reduced in the presence of PE or PI.
authors.affiliation: Kent State University, Department of Chemistry and Biochemistry, PO Box 5190, Kent, OH 44242, USA.
authors.forename: Zachary T
authors.initials: ZT
authors.lastname: Graber
authors.affiliation: Worcester Polytechnic Institute, Department of Chemistry and Biochemistry, 100 Institute Road, Worcester, MA 01605, USA.
authors.forename: Arne
authors.initials: A
authors.lastname: Gericke
authors.affiliation: Kent State University, Department of Biological Sciences, PO Box 5190, Kent, OH 44242, USA. Electronic address: ekooijma@kent.edu.
authors.forename: Edgar E
authors.initials: EE
authors.lastname: Kooijman
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D002784:Cholesterol; D008274:Magnesium; D002118:Calcium
keywords: Lipid domain formation; Lipid electrostatics; Lipid signaling; Model membranes; PI(4,5)P(2); Solid-state (31)P NMR