Patch-clamp coordinated spectroscopy shows P2X2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels

ATP-gated P2X receptors display ion permeability increases within seconds of receptor activation as the channels enter the I-2 state, which is permeable to organic cations and dye molecules. The mechanisms underlying this important behavior are not completely understood. In one model, the I-2 state is thought to be due to opening of Pannexin-1 (Panx-1) channels, and, in the second, it is thought to be an intrinsic P2X property. We tested both models by measuring ion and dye permeability and used a patch-clamp coordinated spectroscopy approach to measure conformational changes. Our data show that Panx-1 channels make no detectable contribution to the P2X(2) receptor I-2 state. However, P2X2 receptors display permeability dynamics, which are correlated with conformational changes in the cytosolic domain remote from the selectivity filter itself. Finally, the data illustrate the utility of a new approach, using tetracysteine tags and biarsenical fluorophores to measure site-specific conformational changes in membrane proteins.