Epithelial Na+ channels (ENaC) coexist with a family of ATP-gated ion channels known as P2X receptors in the renal collecting duct. Although ENaC is itself insensitive to extracellular ATP, tubular perfusion of ATP can modify the activity of ENaC. To investigate a possible regulatory relationship between P2X receptors and ENaC, coexpression studies were performed in Xenopus oocytes. ENaC generated a persistent inward Na+ current that was sensitive to the channel blocker amiloride (I(am-s)). Exogenous ATP transiently activated all cloned isoforms of P2X receptors, which in some cases irreversibly inhibited I(am-s). The degree of inhibition depended on the P2X receptor subtype present. Activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptor subtypes inhibited I(am-s), whereas activation of P2X1, P2X3, and P2X5 receptors had no significant effect. The degree of inhibition of I(am-s) correlated positively with the amount of ionic charge conducted by P2X receptor subtypes. ENaC inhibition required Na+ influx through I(am-s)-inhibiting P2X ion channels but also Ca2+ influx through P2X4 and P2X(4/6) ion channels. P2X-mediated inhibition of I(am-s) was found to be due to retrieval of ENaC from the plasma membrane. Maximum amplitudes of ATP-evoked P2X-mediated currents (I(ATP)) were significantly increased for P2X2, P2X(2/6), and P2X5 receptor subtypes after coexpression of ENaC. The increase in I(ATP) was due to increased levels of plasma membrane-bound P2X receptor protein, suggesting that ENaC modulates protein trafficking. In summary, ENaC was downregulated by the activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptors. Conversely, ENaC increased the plasma membrane expression of P2X2, P2X(2/6), and P2X5 receptors.