P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.

The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327-357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. ytolytic pore formation by extracellular adenosine triphosphate (ATP) was first described in mast cells and was outright suggested to involve the activation and subsequent pore dilatation of a hypothetical ATP receptor channel (1). A similar ATP-triggered pore-forming activity was subsequently found in many immune and inflammatory cells and certain transformed cell lines. The distinctive feature that is unique to this tentatively termed P2Z receptor (2) is that brief application of ATP (in its tetraanionic form, ATP 4− ) evokes depolarizing cation fluxes, whereas prolonged application of ATP 4− causes formation of cytolytic pores with a molecular cutoff of ∼900 Da (for review, see ref.3). The cytolytic activity was eventually assigned to the P2X7 receptor (P2X7R), the seventh and final member of the P2X receptor family. In HEK293 cells, the recombinant P2X7R conferred the same responses that were attributed previously to the P2Z receptor, including the dual-mode operation as a cation channel and a cytolytic pore (4, 5). Today, it is widely believed that P2X7R can mediate apoptotic or necrotic cell death under pathophysiological conditions (4-6).The cytolytic activity of human P2X7R (hP2X7R) has been attributed to a time-dependent dilation of the integral ion channel based on macroscopic current recordings of various cell types (7-11). Particularly revealing was the observation that substituting T348 and D352 with basic residues in the channellining second transmembrane domain (TM2) of the rat P2X7R (rP2X7R) simultaneously increased the permeability of the normally cationic channel for Cl − and an acidic fluorescent dye with an effective diameter of >10 Å (12). A "channel-to-pore" dilatation that changed the permeation characteristic from small inorganic cations (e.g., Na + , K + , and Ca 2+) to large organic cations [e.g., N-methyl-D-glucamine (NMDG + )] has also been found for P2X2, P2X2/X3, and P2X4 receptors (13, 14) and certain transient receptor potential (TRP) channels (15, 16). In contrast, in extended single-chann...

P2X 7 receptors are ATP-gated cation channels composed of three identical subunits, each having intracellular amino and carboxyl termini and two transmembrane segments connected by a large ectodomain. Within the P2X family, P2X 7 subunits are unique in possessing an extended carboxyl tail. We expressed the human P2X 7 subunit as two complementary fragments, a carboxyl tail-truncated receptor channel core (residues 1-436 or 1-505) and a tail extension (residues 434 -595) in Xenopus laevis oocytes. P2X 7 channel core subunits efficiently assembled as homotrimers that appeared abundantly at the oocyte surface, yet produced only ϳ5% of the full-length P2X 7 receptor current. Co-assembly of channel core subunits with full-length P2X 7 subunits inhibited channel current, indicating that the lack of a single carboxyl tail domain is dominant-negative for P2X 7 receptor activity. Co-expression of the tail extension as a discrete protein increased ATPgated current amplitudes of P2X 7 channel cores 10 -20-fold, fully reconstituting the wild type electrophysiological phenotype of the P2X 7 receptor. Chemical cross-linking revealed that the discrete tail extension bound with unity stoichiometry to the carboxyl tail of the P2X 7 channel core. We conclude that a non-covalent association of crucial functional importance exists between the carboxyl tail of the channel core and the tail extension. Using a slightly shorter P2X 7 subunit core and subfragments of the tail extension, this association could be narrowed down to include residues 409 -436 and 434 -494 of the split receptor. Together, these results identify the tail extension as a regulatory gating module, potentially making P2X 7 channel gating sensitive to intracellular regulation. The P2X 7 receptor, an ATP-gated cation channel, is expressed predominantly in immune cells (such as macrophages and lymphocytes), glial cells, and epithelial cells (for recent reviews, see Refs. 1 and 2). Activation of the P2X 7 receptor has been implicated in pivotal inflammatory responses resulting from ATP-stimulated pro-inflammatory cytokine release (particularly of interleukin-1␤ and interleukin-18) through exosomes (3) during cell proliferation and apoptosis (1, 2). These functions of the P2X 7 receptor have been attributed to its unusual dual role as a classic ligand-gated channel for small cations and as a cytolytic pore (4, 5).Cloning of the P2X 7 receptor revealed a typical P2X subunit "core" structure, with a short cytoplasmic NH 2 -terminal tail and two transmembrane segments connected by a large N-glycosylated ectodomain. However, the P2X 7 receptor has a carboxyl tail extension that is 120 -200 amino acids longer than that of the other six P2X family members, P2X 1 -P2X 6 (4, 5). Because the cytolytic pore-forming ability of P2X 7 is not shared by the other P2X receptor subtypes, this function has been plausibly assigned to the long carboxyl tail. Indeed, truncation of most of the extra portion of the carboxyl tail of P2X 7 prevented cytolytic pore formation, apparently without...

Background: Heteromeric P2X receptors increase the diversity of rapid ATP signaling. Results: Non-functional P2X2, P2X3, and P2X6 subunit mutants were used to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. Conclusion:The subunit stoichiometry of P2X2/3 and P2X2/6 is 1:2 and 2:1, respectively. Significance: Recognition sites between P2X2 and its partners rather than random association may govern the subunit composition of the receptor trimers.