Protein kinase A regulation of P2X4 receptors: Requirement for a specific motif in the C-terminus

The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X4R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X4R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X4R expressing HEK-293 cells. To define which region of the P2X4R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X4R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X4R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\ P2X4-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca2+ signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X4R, markedly enhances ATP-stimulated P2X4R currents and hence cytosolic Ca2+ signals. This may occur at least in part, by altering the trafficking of a population of P2X4R present at the plasma membrane.