Abstract
P2X receptors (P2XR) act as ligand-gated, cation-selective ion channels. A common characteristic of all seven P2X family members is a conserved consensus sequence for protein kinase C (PKC)-mediated phosphorylation in the intracellular N-terminus of the receptor. Activation of PKC has been shown to enhance currents through P2X(3)R, however the molecular mechanism of this potentiation has not been elucidated. In the present study we show that activation of PKC can enhance adenosine triphosphate (ATP)-mediated Ca(2+) signals approximately 2.5-fold in a DT-40 3KO cell culture system (P2 receptor null) transiently overexpressing P2X(3)R. ATP-activated cation currents were also directly studied using whole cell patch clamp techniques in HEK-293 cells, a null background for ionotropic P2XR. PKC activation resulted in a approximately 8.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X(3)R cDNA, but had no effect on currents through either P2X(4)R- or P2X(7)R-transfected cells. P2X(3)R-transfected HEK-293 cells were metabolically labeled with (32)PO(4)(-) and following treatment with phorbol-12-myristate-13-acetate (PMA) and subsequent immunoprecipitation, there was no incorporation of (32)PO(4)(-) in bands corresponding to P2X(3)R. Similarly, in vitro phosphorylation experiments, utilizing purified PKC catalytic subunits failed to establish phosphorylation of either P2X(3)R or P2X(3)R-EGFP. These data indicate that PKC activation can enhance both the Ca(2+) signal as well as the cation current through P2X(3)R, however it appears that the regulation is unlikely to be a result of direct phosphorylation of the receptor.