Contribution of transmembrane regions to ATP-gated P2X2 channel permeability dynamics

Abstract

ATP-gated P2X2 channels undergo activation-dependent permeability increases as they proceed from the selective I1 state to the I2 state that is readily permeable to organic cations. There are two main models about how permeability changes may occur. The first proposes that permeability change-competent P2X channels are clustered or redistribute to form such regions in response to ATP. The second proposes that permeability changes occur because of an intrinsic conformational change in P2X channels. In the present study we experimentally tested these views with total internal reflection fluorescence microscopy, electrophysiology, and mutational perturbation analysis. We found no evidence for clusters of P2X2 channels within the plasma membrane or for cluster formation in response to ATP, suggesting that channel clustering is not an obligatory requirement for permeability changes. We next sought to identify determinants of putative intrinsic conformational changes in P2X2 channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I1 state to the dilated I2 state. Initial channel opening to the I1 state was only weakly affected by A1a substitutions, whereas dramatic effects were observed for the higher permeability I2 state. Ten residues appeared to perturb only the I1-I2 transition (Phe31, Arg34, Gln37, Lys53, Ile328, Ile332, Ser340, Gly342, Trp350, Leu352). The data favor the hypothesis that permeability changes occur because of permissive motions at the interface between first and second transmembrane domains of neighboring subunits in pre-existing P2X2 channels.