P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X₇ receptor couples cytoskeletal rearrangements such as membrane blebbing. We used affinity purification of the rat P2X₇ receptor followed by mass spectroscopy and immunoblotting to identify proteins in human embryonic kidney cells that interact with the receptor. We found laminin α₃, integrin β₂, β-actin, α-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase, and the receptor protein tyrosine phosphatase β. Activation of the P2X₇ receptor with the agonist 2Ô,3Ô-benzoyl-(4-benzoyl)ATP resulted in its dephosphorylation on tyrosine; by systematic mutagenesis we identified the residue involved as Tyr³⁴³ in the putative second transmembrane domain. Whole-cell recordings from cells expressing P2X₇ receptors showed that repeated applications of a high concentration of agonist led to a strong decline in the amplitude of the current; this was prevented by phosphatase inhibitors. Phosphatase inhibitors also accelerated membrane blebbing. The results indicate that activation of the P2X₇ receptor results in the stimulation of an associated receptor protein tyrosine phosphatase. Dephosphorylation of the receptor on Tyr³⁴³ inhibits the flow of ionic current and impairs coupling to the downstream effectors leading to the cytoskeleton.