From the initial functional characterization of the permeabilizing “P2Z” ATP receptor 30 years ago (51
), through the molecular identification of the P2Z receptor phenotype as the product of the P2X7R gene (2
), and up to the most recent analyses of in vivo
functional deficits in P2X7-knockout mice (10
), two fundamental and perplexing questions regarding the P2X7R have been repeatedly considered. First, why does this particular ATP receptor, in sharp contrast to the six other P2X receptor subtypes, require millimolar levels of extracellular ATP for activation when studied in isolated cells? This unusual characteristic suggests that low affinity variants of an ancestral P2X7R were favored by positive selection as the receptor acquired its physiological roles as a regulator of proinflammatory signaling and cell death. Low ATP affinity prevents inadvertent activation of these highly consequential but poorly reversible responses until leukocytes accumulate at sites of tissue damage or microbial invasion. However, this raises a second and corollary question: how does the P2X7R become activated in leukocytes within these latter tissue compartments given the receptor’s low affinity of ATP? Recent studies support three possible mechanisms that are not mutually exclusive: 1) highly localized accumulation of ATP for autocrine activation of P2X7R within diffusion-restricted cell surface compartments (13
); 2) allosteric modulation of ATP affinity via conformational changes in P2X7 trimeric channels produced by local biophysical conditions or covalent modification of the P2X7R protein itself (15
); and 3) the ATP-independent activation of P2X7R via conformational changes produced by ADP-ribosylation of key arginines within the extracellular loop of the P2X7R (20
). The experiments described in this report provide new insights into the latter two regulatory mechanisms and additionally suggest that a fourth mechanism – involving tissue/cell-selective expression of accessory molecules and/or of P2X7R splice variants – contribute to the regulation of P2X7R function.
The ability of NAD to drive the covalent modification of extracellular residues of the P2X7R comprises a novel mechanism to produce relatively long-lasting changes in the conformational state of these ligand-gated ion channels during transient increases in extracellular NAD, ATP, and other normally intracellular metabolites, such as lyso-lipids, that can regulate P2X7R function (22
). Previous studies demonstrated that NAD can trigger P2X7R activation in murine T lymphocytes even when these cells were incubated in the presence of apyrase to scavenge any released ATP (20
). Moreover, P2X7R activation was sustained in T cells briefly treated with NAD and then washed free of this nucleotide. In contrast, ATP-stimulated P2X7R rapidly deactivated when T cells were transferred to ATP-free medium. These findings indicate that ADP-ribosylation of P2X7R subunits in murine T cells induces a conformational change sufficient to gate the opening of these trimeric channels even in absence of ATP binding. However, our studies of P2X7R function in murine macrophages and HEK293 cells indicate that this ATP-independent activation of P2X7R by ADP-ribosylation is not a general mode of P2X7R regulation but rather reflects specialized conditions present in murine T lymphocytes.
Notably, while NAD by itself was able to gate P2X7R in NZW T lymphocytes expressing solely ART2.1, it failed to activate P2X7R either in murine macrophages that co-express native P2XR7 and ART2.1 or in HEK293 cells engineered to co-express murine P2X7R and murine ART2 ecto-enzymes. However, NAD acted synergistically with ATP to regulate P2X7R in both the macrophages and the engineered HEK cells, and this effect of NAD was strictly dependent on the expression of ART2.1 or ART2.2 in both cell models. How can these regulatory effects of NAD/ART2 on ATP-dependent P2X7R activation observed in murine macrophages and HEK293 cells be reconciled with the robust ATP-independent activation by NAD/ART2 in murine T cells? A possible explanations for the difference in P2X7R signaling observed between myeloid and lymphoid cells are that T cells, but not macrophages or HEK cells, express other regulatory proteins that facilitate the ATP-independent gating of P2X7R in response to ADP-ribosylation, or that the local membrane microenvironments containing P2X7R and ART2 are different in the two cell types. Another possible explanation might be the expression of different recently identified splice variants of rodent P2X7R in T cells versus macrophages. Indeed, Taylor et al. have recently reported that P2X7 receptor function is preserved in the T lymphocytes, but not macrophages, from one strain of P2X7-null mice which was generated by lacZ insertion into exon 1 of the p2rx7
). Determining whether different murine tissues, particularly hematopoietic cells, differentially express splice variants of the P2X7R with altered functional responses to ART2-mediated modification is an important goal for future experiments.
It is important to consider how ADP-ribosylation of key Arg residues may affect the conformation of these trimeric channels. Electrophysiological analyses of P2X-family channels, at the whole cell and single-channel levels, indicate that at least 2, and probably 3, molecules of ATP need to be bound per channel for optimal gating (9
). Moreover, the critical ATP binding sites appear to be formed at the interfaces between the extracellular loops of individual subunits, rather than within each subunit loop as initially hypothesized (62
). In this regard, the covalently associated ADP-ribose at Arg-125 of a P2X7 subunit may interact with the key interfacial amino residues that form the ATP-binding site. However, ADP-ribose is larger than ATP per se and it is unclear whether the P2X7R channel complex can accommodate ADP-ribosylation of all 3 subunits or whether only 1 or 2 subunits per channel can be efficiently modified. Differences in the number of covalently modified subunits per channel, due possibly to steric hindrance, may underlie the distinctive consequences of NAD/ART2 action on P2X7R function in macrophages versus T cells. ADP-ribosylation of these receptors in macrophages may be limited to only 1 or 2 subunits per channel, which is insufficient for gating but sufficient for positive allosteric action at the remaining interfacial ATP binding sites. This would be consistent with the observed increase in potency of ATP at P2X7R in ART2.1 expressing macrophages (or HEK293 cells) pretreated with NAD. In contrast, the predominant P2X7R channels in T cells, or the mutant P2X7R-R276K channels in HEK cells, may have conformations that accommodate permit ADP-ribosylation of all 3 subunits.
It is currently unclear whether ADP-ribosylation is a common mechanism for the activation or sensitization of P2X7R signaling in other tissues or organisms. Notably, the Arg-125 and Arg-133 residues are conserved in the human P2X7R (64
), but the human ART2A and ART2B are transcriptionally silent pseudo-genes (26
). Thus, human T cells and macrophages lack the capacity for cis
-regulation of P2X7R by a co-expressed ecto-ART. However, human ART1 is constitutively expressed in neutrophils and this GPI-anchored enzyme is rapidly shed during neutrophil activation in response to bacterial infection (66
). ART1 is also expressed by human airway epithelial cells basally and at increased levels in response to bacterial mediators (67
). Thus, the P2X7R in human macrophages and T cells might be trans
-regulated by shed ART1 that accumulates at sites of bacterial infection and neutrophil recruitment. Such a mechanism may also be operative in mice which also express ART1 in other tissues such as cardiac and skeletal muscle (70
NAD is released to extracellular environments during the early stage of inflammatory response (58
). Besides its ability to trigger P2X7R-dependent T cell death (20
), extracellular NAD has been reported as an agonist for P2Y11 receptors in human granulocytes (48
). Our study now shows that NAD also increases the sensitivity of the P2X7R to ATP gating in macrophages. This action of NAD requires expression of the thiol-sensitive ART2.1 enzymes and reduced thiols, such as glutathione and cysteine, that can accumulate at inflammatory loci due to release from activated macrophages and the hypoxia that often characterizes such loci (31
). We have found that ART2.1 is widely expressed in other leukocytes, such as dendritic cells and B lymphocytes (71
). Thus, the sensitization of ATP-dependent P2X7R activation by NAD/ART2.1 may provide an additional layer of regulatory control in multiple phases of innate and adaptive immunity.