The molecular motions that give rise to permeability changes in ATP-gated P2X2 channels are not fully understood (Khakh et al., 1999; Virginio et al., 1999; Eickhorst etal., 2001). We extended work presented previously (Fisher et al., SFN 2003), to show that fusion of the plasma membrane binding pleckstrin homology (PH) domain of phospholipase C to the C termini of P2X2 channels resulted in a splayed conformation of the cytosolic domain, such that the FRET efficiency between CFP and YFP attached to it is reduced from 35±5% to 17±2%. Interestingly, this splayed conformation was not permissive for permeability changes (0.2±0.04 v 2.8±0.4-fold increase in pNMDG+/pNa+ for PH tagged and wt P2X2), or for ATP-evoked changes in FRET measured between fluorophores on the C termini (-1.9±1.8% v –14.1±2.0% change in FY/FC for PH-tagged and wt P2X2). ATP-evoked changes in permeability (2.8±0.8-fold increased pNMDG+/pNa+) and FY/FC (-9.3±1.8% change) were restored for P2X2-PH channels when PIP2 was cleaved to un-tether ...