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 PLCδ1-PH dissociation kinetics can be distinguished by their effective k off values determined from fitting fluorescence traces to a single exponential equation. Our measurements for the k off of AKT-PH-GFP and PLCδ1-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 PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP 3 ) to samples during the unroofing process, we measured a much faster k off of 1.54 ± 0.42 s −1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly depressed through competitive action by IP 3 for the same PH domain binding site as phosphatidylinositol (4,5)-bisphosphate (PIP 2 ). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP 2 levels modulate the interaction between membrane associated proteins and the plasma membrane.