Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling

Phosphatidylinositol 4,5-bisphosphate (PIP₂) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP₂ continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP₂ molecules in the top leaflet are fully mobile, while the PIP₂ molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP₂ species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP₂ flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H₂O to D₂O, it was shown that the primary interaction between PIP₂ and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP₂. Moreover, PIP₂ immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP₂ on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.