Phosphatidylinositol phosphate binding domains exhibit complex dissociation properties at the inner leaflet of plasma membrane sheets

The pleckstrin homology (PH) domain is a lipid targeting motif that binds with high specificity to phosphatidylinositol phosphate (PIP) lipids. Using TIRF microscopy, we followed the dissociation of GFP-tagged PH domains from the plasma membranes of rapidly unroofed cells and found that AKT-PH and PLC{delta}1-PH dissociation kinetics can be distinguished by their effective koff values determined from fitting fluorescence traces to a single exponential equation. Our measurements for the koff of AKT-PH-GFP and PLC{delta}1-PH-GFP were significantly different (p < 0.05) at 0.39 {+/-} 0.05 s-1 and 0.56 {+/-} 0.17 s-1, respectively. Furthermore, we identified substantial rebinding events in our measurements of PLC{delta}1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much faster koff of 1.54 {+/-} 0.42 s-1 for PLC{delta}1-PH-GFP, indicating that rebinding events are significantly depressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol (4,5)-bisphosphate (PIP2). We discuss the complex character of our PLC{delta}1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane associated proteins and the plasma membrane.