Mammalian ATP-gated nonselective cation channels (P2X) exist as seven different subtypes, each containing a large ectodomain, two transmembrane domains, and intracellular N and C termini. As a unique structural feature, P2X₇ has a long C-terminus which is thought to operate as a platform for various protein interactions. P2X₇ is important for pain perception and has a role in immunity, inflammation and during apoptosis. Opening of P2X₇ by millimolar concentrations of ATP leads to instantaneous activation of a nonselective cation conductance and a delayed activation of a nonselective large conductance pore. Because previous data on activation of P2X₇ in Xenopus oocytes have been controversial, we expressed human P2X₇ in oocytes and examined activation of currents by 1 mM ATP. As expected application of 1 mM ATP rapidly stimulated an inward current that was inhibited by both the competitive ATP-antagonist AZ11645373 (50 nM) and by the P2X₇ pore blocker 5-(N, N-hexamethylene)-amiloride (40 mM). Moreover, with a delay of about 2 min, a very large whole cell current was activated by 1 mM ATP that was only slightly inhibited when extracellular Cl^- and Na⁺ were replaced by gluconate and NMDG⁺, respectively. Furthermore activation of the pore current was only slightly inhibited by chelating Ca²⁺ with BAPTA-AM (50 mM), but was largely reduced in the absence of extracellular Ca²⁺. These data suggest that influx of Ca²⁺ through P2X₇ is important for activation of the large nonselective pore. A point mutation within the C-terminus of P2X₇ (E496A) has been demonstrated to inhibit activation of the pore in mammalian cells. In deed when we expressed P2X₇-E496A in Xenopus oocytes, activation of the pore was also largely reduced. Notably, pore currents activated in Xenopus oocytes were largely inhibited by typical Cl^- channel blockers such as NPPB or tannic acid. Taken together, these data indicate that activation of a large conductance pore during stimulation of P2X₇ can be observed in Xenopus oocytes. Local increase in intracellular Ca²⁺ as well as an intact putative site for protein interaction within the C-terminus of P2X₇ are required for complete activation of the pore. We propose that activation of an unknown independent membrane protein is necessary for pore formation induced by P2X₇ purinoreceptors.

Work supported by DFG SFB699-A7. We thank Prof. G. Schmalzing for supplying the cDNA for the P2X₇ receptor and Ms. E. Tartler for excellent technical assistance