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...

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum.

P2X7 receptors are ATP-activated cation channels involved in inflammatory and immunological reactions to cell injury. Their second transmembrane domain (TM2) is believed to line the ion channel pore. Here, we applied cysteine scanning to investigate the role of TM2 residues in the permeation and gating of the human P2X7 receptor (hP2X7R). Following heterologous expression of the hP2X7R cysteine substitution mutants in Xenopus oocytes, their function was measured by voltage-clamp in the whole-cell mode and by single-channel voltage-clamp in the outside-out configuration before and after application of methanethiosulfonate (MTS) reagents, which can covalently react with the substituted cysteines. Furthermore, we checked if the hP2X7R cysteine substitution mutants expressed in Xenopus oocytes could be labeled by extracellularly applied sulfo-Cy5-maleimide. At the putative extracellular end of the hP2X7R TM2 domain, we found cysteine substitution mutants which could be labeled by the fluorescent Cy5 dye, but the ATP-induced currents were not affected by MTS administration. This indicates that the corresponding amino acid residues are extracellularly accessible, but are neither involved in the gating nor in the permeation of the ion channel. One hP2X7R mutant (S342C) could be Cy5-labeled and the ATP-induced current was blocked by the cationic 2-aminoethyl methanethiosulfonate (MTSEA) pointing to an involvement of S342 in ion permeation or to a stabilization of the closed state of the channel. Several other cysteine mutants were dye labeled and exhibited increased ATP-induced currents after MTSEA application. This MTSEA-dependent current increase was due to an increase of the channel's open probability indicating that the corresponding amino acid residues are involved in in P2X7R channel gating. The structure-function relationship of the TM2 domain of the hP2X7R is discussed using a hP2X7R homology model based on the published X-ray structures of the zebrafish-P2X4 receptor.

P2X4 and P2X7 are members of the P2X receptor family, comprising seven isoforms (P2X1-P2X7) that form homo-and heterotrimeric non-specific cation channels gated by extracellular ATP. P2X4 and P2X7 are widely coexpressed, particularly in secretory epithelial cells and immune and inflammatory cells, and regulate inflammation and nociception. Although functional heteromerization has been established for P2X2 and P2X3 subunits expressed in sensory neurons, there are contradictory reports regarding a functional interaction between P2X4 and P2X7 subunits. To resolve this issue, we coexpressed P2X4 and P2X7 receptor subunits labeled with green (EGFP) and red (TagRFP) fluorescent proteins in Xenopus laevis oocytes and investigated a putative physical interaction between the fusion proteins by Förster resonance energy transfer (FRET). Coexpression of P2X4 and P2X7 subunits with EGFP and TagRFP located in the extracellular receptor domains led to significant FRET signals. Significant FRET signals were also measured between C-terminally fluorophore-labeled full-length P2X4 1−384 and C-terminally truncated fluorescent P2X7 1−408 subunits. We furthermore used the two-electrode voltage clamp technique to investigate whether human P2X4 and P2X7 receptors (hP2X4, hP2X7) functionally interact at the level of ATP-induced whole-cell currents. Concentration-response curves and effects of ivermectin (P2X4-potentiating drug) or BzATP (P2X7-specific agonist) were consistent with a model in which coexpressed hP2X4 and hP2X7 do not interact. Similarly, the effect of adding specific inhibitors of P2X4 (PSB-15417) or P2X7 (oATP, A438079) could be explained by a model in which only homomers exist, and that these are blocked by the respective antagonist. In conclusion, we show that P2X4 and P2X7 subunits can form heterotrimeric P2X4/P2X7 receptors. However, unlike observations for P2X2 and P2X3, coexpression of P2X4 and P2X7 subunits does not result in a novel electrophysiologically discriminable P2X receptor phenotype.