ATP-induced internalization of amphibian epithelial P2X receptors is linked to channel opening

Abstract.

A family of ATP-gated, non-selective cation channels known as P2X receptors are involved in purinergic synaptic transmission and other, as yet poorly defined, functions. After repeated exposure to ATP some of these channels no longer respond, a phenomenon known as run-down. In some P2X receptor subtypes, run-down has been associated with endocytosis. Recently, we have cloned a P2X receptor from larval amphibian skin (fP2X5) that shows profound run-down. To visualize the receptor in the plasma membrane of Xenopus oocytes, it was fused to a green fluorescent protein (GFP). Localized membrane fluorescence was observed in fP2X5 GFP-injected oocytes. Typical voltage-clamp current run-down was observed in response to repeated ATP exposure, indicating that GFP did not alter channel function. Membrane fluorescence decreased after ATP exposure. The purinergic receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blocked ATP-induced membrane fluorescent decreases and voltage-clamp currents. A T18A-fP2X5 mutant showed no ATP-activated currents when expressed in oocytes. GFP fusion constructs of this mutant showed membrane fluorescence, indicating insertion of a non-functional channel. In contrast to the wild-type fP2X5, ATP caused no decrease in membrane fluorescence of the mutant channel. Taken together these data suggest that channel opening is necessary for receptor internalization.