Interaction of Phosphatidylinositol-4,5-Bisphosphate with Potential Clustering Agents Ca2+, Mg2+, and Cholesterol

Abstract

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is an important signaling lipid in the cell plasma membrane, playing an important role in many diverse signaling processes. It is important to gain an understanding of how PI(4,5)P2's role in these signaling processes is regulated. For example, it has been proposed that regulation by PI(4,5)P2 is based on its lateral localization within the plasma membrane, with multiple pools of PI(4,5)P2 used for different signaling purposes. In vitro studies have indicated that both Ca2+ and cholesterol have the capacity to promote formation of PI(4,5)P2 clusters in model membranes. To shed light on this we have examined the interaction of PI(4,5)P2 with Ca2+, Mg2+, and cholesterol using solid-state MAS 31P NMR. The solid state 31P-NMR allows us to examine the differential effects of Ca2+, Mg2+, and cholesterol on the 4 and 5 phosphomonesters of PI(4,5)P2 independently. We examined phosphatidylcholine multilamellar vesicles containing near physiological concentration of PI(4,5)P2 in the presence of micro and millimolar concentrations of Ca2+ and Mg2+. The 4- and 5-phosphates of PI(4,5)P2 were both found to shift downfield in the presence of Ca2+ and Mg2+, indicating increased deprotonation of PI(4,5)P2 due to association of the cations. This effect was significantly larger in the presence of Ca2+. Multilamellar vesicles containing PC and PI(4,5)P2 with varying amounts of cholesterol were also studied. The 4- and 5-phosphates of PI(4,5)P2 were found to have a significant downfield shift in the presence of 40 mol% cholesterol. The cumulative effects of cholesterol in combination with the common inner leaflet phospholipids, PE and PI, were also examined.