Many effects of extracellular ATP on cells of the immune system have been attributed to the presence of a so-called P2Z receptor. Recent work has shown that one of the members of the P2X family of ATP-gated receptors Soto et al. 1997;Ralevic & Burnstock, 1998;MacKenzie et al. 1999), designated P2X 7 , shares many phenotypical properties with the P2Z receptor upon heterologous expression, suggesting that the two are identical. The P2X 7 receptor is therefore also referred to as P2Z/P2X 7 receptor (Di Virgilio, 1995;Di Virgilio et al. 1998). A peculiarity of the P2X 7 subunit is its very long intracellular C-terminal tail, which is 196 or 132 amino acids longer than that of the P2X 1 or P2X 2 subunit isoform, respectively.Several studies attempting to characterise the recombinant P2X 7 receptor have provided information about its complex function, which is not yet fully understood. During short applications of ATP lasting a few seconds, the P2X 7 receptor behaves like a typical P2X family member, exhibiting permeability to small cations only. However, upon prolonged or repeated applications of ATP, large non-selective pores are formed in the plasma membrane of some cells expressing P2X 7 (Surprenant et al. 1996;Rassendren et al. 1997;Virginio et al. 1999), which have been attributed to the receptor itself. On the other hand, it has also been suggested that the pores represent distinct entities, which become activated subsequent to the stimulation of P2X 7 receptors (Coutinho-Silva & Persechini, 1997;Schilling et al. 1999).In native cells, ATP elicits different effects over a wide concentration range. For instance, ATP increases the cell 1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X 7 receptor (hP2X 7 ), an N-terminally hexahistidyl-tagged hP2X 7 receptor (His-hP2X 7 ), or a truncated His-hP2X 7 receptor (His-hP2X 7 ∆C) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique.2. The activation time course of the wild-type hP2X 7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component.3. The amplitude of the exponentially activating current component of the wild-type hP2X 7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity noncooperative activation sites with apparent dissociation constants of about 4 and 200 µM free ATP 4_ , respectively.4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 µM.5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X 7 or His-hP2X 7 ∆C.6. Our data indicate that the hP2X 7 receptor possesses at least two types of activation sites, which differ in ATP 4_ sensitivity by a factor of 50. The degree of occupatio...

Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.

1. Using the patch clamp method in the outside-out configuration, purinoceptor-dependent unitary currents were measured in tonsillar and transformed tonsillar human B lymphocytes. 2. Single channel currents were evoked by ATP4-, the free-acid form of ATP, and by 2',3'-Obenzoyl-4-benzoyl-ATP (BzATP) in the micromolar concentration range, but not by 10 mM ADP3-or 05 mM Mg2+-bound ATP. 3. The channels could be activated and deactivated several times for as long as 30 min even in the absence of intracellular ATP, GTP, or glucose.4. The channels were selective for small cations and had a conductance of 9 pS with Cs' as the intracellular and Nae as the extracellular monovalent cation.5. The half-maximal activation of the channels was obtained by 114 /M ATP4-and by 16 /M BzATP. The increase in the open probability after raising the ATP4-concentration was mainly due to a decrease in the times the channels spend in the closed state. 6. It is concluded that human B lymphocytes possess cationic channels directly gated by extracellular ATP4-. Their agonist binding characteristics are typical for P2Z purinoceptors, but their permeation behaviour is different from the large non-specific pores formed by ATP4-in fibroblasts, macrophages and mast cells.Receptors for the extracellular purines ATP and ADP are classified as P2 purinoceptors. Whereas the subtypes P2T, P2U and P2Y are G-protein-coupled receptors, the subtype P2X is a ligand-gated ionic channel (Bean, 1992;Dubyak & El-Moatassim, 1993;Harden, Boyer & Nicholas, 1995;.The P2Z receptor is found in cells of the immune and inflammatory system. It is characterized by binding of only free uncomplexed ATP4-, in the concentration range of about 100 /SM, and by the absence of desensitization. In

Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of Ip.

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.

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 P2X41-384 and C-terminally truncated fluorescent P2X71-408 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.

We report the activation of outwardly rectifying anion currents by sphingosine-1-phosphate (S1P) in the murine macrophage cell line RAW 264.7. The S1P-induced current is mainly carried by anions, because the reversal potential of the current was shifted by replacement of extracellular Cl(-) by glutamate(-) but not when extracellular Na(+) was substituted by Tris(+). The inhibition of the current by hypertonic extracellular or hypotonic intracellular solution as well as the inhibitory effects of NPPB, tamoxifen, and glibenclamide indicates that the anion current is mediated by volume-regulated anion channels (VRAC). The S1P effect was blocked by intracellular GDPβS and W123, which points to signaling via the S1P receptor 1 (S1PR1) and G proteins. As cytochalasin D diminished the action of S1P, we conclude that the actin cytoskeleton is involved in the stimulation of VRAC. S1P and hypotonic extracellular solution induced secretion of ATP from the macrophages, which in both cases was blocked in a similar way by typical VRAC blockers. We suppose that the S1P-induced ATP secretion in macrophages via activation of VRAC constitutes a functional link between sphingolipid and purinergic signaling in essential processes such as inflammation and migration of leukocytes as well as phagocytosis and the killing of intracellular bacteria.

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