Thermal stability of bivalent cation/phosphoinositide domains in model membranes

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂ and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P₃) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg²⁺ ions. While PI(4)P shows in the presence of Ca²⁺ no clustering, PI(4,5)P₂ forms with Ca²⁺ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P₃ with Ca²⁺ is less than what was observed for PI(4,5)P₂, yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg²⁺ or Ca²⁺ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca²⁺ with PI(4,5)P₂. Also, in the presence of cholesterol, the interaction of Mg²⁺ with PI(4,5)P₂ remains weak. PI(3,4,5)P₃ does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca²⁺ and Mg²⁺ was not influenced by the presence of cholesterol.