Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.

There are seven P2X receptor cDNAs currently known. Six homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5) P2X receptor channels have been characterized in heterologous expression systems. Homomeric P2X1 and P2X3 receptors are readily distinguishable by their rapid desensitization, the agonist action of alpha beta methyleneATP, and the block by 2',3'-O-(2,4,6-trinitrophenyl)-ATP. P2X2 receptors are unique among homomeric forms in their potentiation by low pH. Homomeric P2X4 receptors are much less sensitive to antagonism by suramin and pyridoxal 5-phosphate-6-azo-2',4'-disulfonic acid. Homomeric P2X7 receptors are the only form in which 2',3'-O-(4-benzoylbenzoyl)-ATP is more potent than ATP. The heteromeric P2X2/P2X3 receptor resembles P2X2 in slow desensitization kinetics and potentiation by low pH and is similar to P2X3 with respect to agonism by alpha beta methyleneATP and block by 2',3'-O-(2,4,6-trinitrophenyl)-ATP. Other agonists, antagonists, and ions that can be used to differentiate among the receptors are discussed.

There are few antagonists selective for subtypes of the several P2X receptors, but these are needed to identify the receptors expressed on native cells and tissues. In particular, P2X(4) and P2X(7) receptor subunits are colocalized on immune, epithelial, and exocrine gland cells, but both are relatively insensitive to suramin and pyridoxal-5-phosphate-6-azo-2',4'-disulfonic acid derivative. In this article, we show that Coomassie Brilliant Blue G selectively inhibits P2X(7) receptors with nanomolar affinity. We measured currents in response to P2X receptor activation in HEK293 cells heterologously expressing human or rat P2X(1), P2X(2), P2X(3), P2X(2/3), P2X(4), P2X(1/5), and P2X(7) receptors. Brilliant Blue G produced a noncompetitive inhibition of rat and human P2X(7) receptors with IC(50) values of 10 and 200 nM, respectively. IC(50) values for inhibition of the other receptors ranged from 2 to >30 microM; the rat and human P2X(4) receptors showed IC(50) values of >10 and 3.2 microM. Coomassie Blue G also blocked YO-PRO1 uptake and membrane blebbing, which are uniquely associated with activation of P2X(7) receptors. Thus, Brilliant Blue G is at least 1000-fold more potent at rat P2X(7) receptors than at rat P2X(4) receptors.

P2X receptors are ATP-gated ion channels in the plasma membrane, but activation of the P2X 7 receptor also leads to rapid cytoskeletal re-arrangements such as membrane blebbing. We identi®ed 11 proteins in human embryonic kidney cells that interact with the rat P2X 7 receptor, by af®nity puri®cation followed by mass spectroscopy and immunoblotting [laminin a3, integrin b2, b-actin, a-actinin, supervillin, MAGuK, three heat shock proteins, phosphatidylinositol 4-kinase and receptor protein tyrosine phosphatase-b (RPTPb)]. Activation of the P2X 7 receptor resulted in its dephosphorylation. Whole-cell recordings from cells expressing P2X 7 receptors showed that this markedly reduced subsequent ionic currents and it also slowed membrane bleb formation. By mutagenesis, we identi®ed Tyr 343 in the putative second transmembrane domain as the site of phosphorylation. Thus, we have identi®ed a P2X 7 receptor signalling complex, some members of which may initiate cytoskeletal rearrangements following receptor activation. Others, such as RPTPb, might exert feedback control of the channel itself through its dephosphorylation. Keywords: ion channel/P2X receptors/receptor protein tyrosine phosphatase-b/signalling complex/tyrosine phosphorylation Introduction P2X receptors form a family of ATP-gated ion channels extensively distributed throughout the cells of vertebrates. They are ligand-gated ion channels, each with distinct pharmacological and/or physiological properties. They form as homomers and/or heteromers, and current biochemical evidence suggests that the channel has three or perhaps six subunits (Nicke et al., 1998; North and Surprenant, 2000). The P2X 7 receptor subunit has several features that set it apart from other members of the family (Surprenant et al., 1996). Co-immunoprecipitation experiments indicate that it is the only subunit that does not heteropolymerize with other P2X subunits (Torres et al., 1999). It is primarily localized to epithelia and immune cells, particularly antigen-presenting cells Mutini et al., 1999). Receptor activation requires concentrations of ATP that are 10±100 times higher than those required to activate other P2X receptors, but the agonist af®nity and maximum response can also be modulated 5-to 100-fold by alterations in external monovalent and divalent cations (Surprenant et al., 1996;Rassendren et al., 1997;Michel et al., 1999;Gudipaty et al., 2001). The ionic currents through P2X 7 receptors show considerable plasticity, in the sense that repeated applications of the agonist result in prominent changes in the amplitude and time course of the current elicited by subsequent applications (Surprenant et al., 1996;Rassendren et al., 1997;Hibell et al., 2000).Activation of native ATP receptors in macrophages and macrophage-like cell lines, in which P2X 7 subunits are predominately expressed, is fundamentally different from that observed for other ion channels because it initiates several cellular consequences further downstream. These include alterations in the cell morphology (Cohn...

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.