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.

Regulation of ion transport via apical purinergic receptors in intact rabbit airway epithelium

“…The Ca 2+ i -and cAMPindependent effect of ATP that we ascribe to P2X stimulation was brief, indicating little importance in regulation of epithelial ion transport. The short duration of the P2X response may be due to an internalization of the receptor after binding of the agonist [19]. Thus, the small, initial, positive spike in I sc in response to ATP (Fig.…”

Regulation of Human Recombinant P2X3Receptors by Ecto-Protein Kinase C

“…The N-terminal phosphorylation of P2X receptors may, on the one hand, enhance the rate of desensitization and thereby also modulate the peak current amplitude [P2X 2 (Boue-Grabot et al, 2000)], and, on the other hand, it may decrease the membrane expression of this receptor by interfering with its internalization [P2X 5 (Jensik and Thomas, 2002)]. It has been hypothesized that the completely conserved PKC phosphorylation site of the P2X 3 signaling peptide within the subsequence 1-44 is essential to pass the endoplasmatic reticulum or the plasma membrane (Mager et al, 2004).…”

The h-P2X3 glycoprotein receptor as an example of integrating bioinformatics and structural research

“…PKC1 does not interact with other peptide modules, but is a part of the signalling peptide (fragment 1 -44, the cleavage site is between position 44 and 45). The T18 of P2X5 is homologous with the T12 of P2X3 and it was shown that the T18A mutant blocks P2X5 internalisation and did not show ATP-activated currents [24]. For personal use only.…”

“…In homology to the results of other proteins, it may be speculated that an agonist-promoted downregulation of P2X receptors [23][24][25][26][27][28] is also related to certain N-glycosylation sites. The N-glycosylation site (Asn187) of the β 2 -adrenergic receptor contributes to the internalisation during a long-term agonist exposure and the mutant Asn187Asp failed to display long-term agonist-promoted downregulation [29].…”