Contribution of Transmembrane Regions to ATP-gated P2X2 Channel Permeability Dynamics

Baljit S Khakh,Terrance M Egan

doi:10.1074/jbc.m411324200

Abstract

ATP-gated P2X(2) channels undergo activation-dependent permeability increases as they proceed from the selective I(1) state to the I(2) 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 P2X(2) 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 P2X(2) channels by mapping the transmembrane domain regions involved in the transition from the relatively selective I(1) state to the dilated I(2) state. Initial channel opening to the I(1) state was only weakly affected by Ala substitutions, whereas dramatic effects were observed for the higher permeability I(2) state. Ten residues appeared to perturb only the I(1)-I(2) transition (Phe(31), Arg(34), Gln(37), Lys(53), Ile(328), Ile(332), Ser(340), Gly(342), Trp(350), Leu(352)). 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 P2X(2) channels.

Highlights

ATP-gated P2X channels are a large family of transmittergated cation channels [1,2,3]
P2X2, P2X4, and P2X7 channels display permeability dynamics, whereby the channel pore dilates in an ATP activation- and time-dependent manner from a fairly selective I1 state to the I2 state that is permeable to organic cations (16 –24)
We studied homomeric P2X2 channels because they show robust permeability changes in all HEK cells, they can be labeled innocuously with GFP variants [18, 34], and they likely underlie the high permeability to organic cations measured for natively expressed P2X channels in neurons [16, 17, 19, 27]

Summary

Introduction

ATP-gated P2X channels are a large family of transmittergated cation channels [1,2,3]. Overall the data are consistent with the gating model and the hypothesis that permeability changes occur because of spatially diverse, and perhaps extensive, molecular rearrangements in the transmembrane, pore, and cytosolic domains [34] of pre-existing and stably expressed P2X2 channels.

Results

Conclusion