Annmarie Surprenant

Signaling at Purinergic P2X Receptors

P2X receptors are membrane cation channels gated by extracellular ATP. Seven P2X receptor subunits (P2X(1-7)) are widely distributed in excitable and nonexcitable cells of vertebrates. They play key roles in inter alia afferent signaling (including pain), regulation of renal blood flow, vascular endothelium, and inflammatory responses. We summarize the evidence for these and other roles, emphasizing experimental work with selective receptor antagonists or with knockout mice. The receptors are trimeric membrane proteins: Studies of the biophysical properties of mutated subunits expressed in heterologous cells have indicated parts of the subunits involved in ATP binding, ion permeation (including calcium permeability), and membrane trafficking. We review our current understanding of the molecular properties of P2X receptors, including how this understanding is informed by the identification of distantly related P2X receptors in simple eukaryotes.

P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage.

The proinflammatory IL-1 cytokines IL-1α, IL-1β, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X7 receptors (P2X7R) by extracellular ATP is a key physiological inducer of rapid IL-1β release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X7R−/− mice and found that release of IL-1α, IL-18, as well as IL-1β, by ATP resulted exclusively from activation of P2X7R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1β release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1β independently of panx1 but do not release mature IL-1β because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1β that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1β that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1β.

Nanoparticles can cause DNA damage across a cellular barrier

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

Dynamics of macrophage polarization reveal new mechanism to inhibit IL‐1β release through pyrophosphates

In acute inflammation, extracellular ATP activates P2X7 ion channel receptors (P2X7R) on M1 polarized macrophages to release pro‐inflammatory IL‐1β through activation of the caspase‐1/nucleotide‐binding domain and leucine‐rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X7R on these macrophages nor mechanisms by which macrophages switch from pro‐inflammatory to anti‐inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL‐1β remains high and the inflammasome is intact, P2X7R activation selectively uncouples to the NLRP3‐inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL‐1β release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments.

P2X7 Receptor-Mediated Release of Cathepsins from Macrophages Is a Cytokine-Independent Mechanism Potentially Involved in Joint Diseases

The ATP-gated P2X7 receptor (P2X7R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X7R antagonists are considered to result from inhibition of P2X7R-induced release of proinflammatory cytokines from activated macrophages. However, P2X7Rs are also expressed in resting macrophages, suggesting that P2X7R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X7R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1β and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X7R antagonists, absent from P2X7R−/− mouse macrophages, and not associated with cell death. Our results suggest P2X7R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.

Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions.

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptors sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation

ATP‐gated P2X4 receptors (P2X4R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X4R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X4R protein expression by Western blot and biotinylation assays and functional expression by whole‐cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X4R expression by 2‐ to 7‐fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN‐γ and TNF‐α or IFN‐γ and LPS reduced surface and functional P2X4R expression by 3‐fold without altering total P2X4R protein levels. Alternative activation with IL‐4 or IL‐13 did not alter total, surface or functional expression of P2X4R. This is the first study of the regulation of P2X4R in macrophages by physiological stimuli and presents a picture whereby P2X4R become functional in response to initial phagocytic stimuli but return to a non‐functional state during sustained activation by classical macrophage activation.

Identification of the amino acid residues in the extracellular domain of rat P2X7 receptor involved in functional inhibition by acidic pH

Background and purpose:  P2X7 receptors are potently inhibited by extracellular acidification. The underlying molecular basis remains unknown. This study aimed to examine the role of extracellular histidine, lysine, aspartic acid and glutamic acid residues in the functional inhibition of rat P2X7 receptors by acidic pH.

Identification of an intracellular microdomain of the P2X7 receptor that is crucial in basolateral membrane targeting in epithelial cells.

MINT‐8055849:Beta‐catenin (uniprotkb:B6V8E6) and P2X7R (uniprotkb:Q64663) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Identification of Thr(283) as a key determinant of P2X(7) receptor function

The ATP‐gated P2X7 receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X7 receptor mutations.