The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.DOI: http://dx.doi.org/10.7554/eLife.22153.001

Immune complex-mediated inflammation is a common mechanism of various autoimmune diseases. Glomerulonephritis (GN) is one of these diseases, and the main mechanism of the induction of GN has been unclear. We examined the contribution of Fc receptors in the induction of nephrotoxic GN by establishing and analyzing mice deficient in the Fc receptor ␥ chain (FcR ␥ ). Whereas all wild-type mice died from severe glomerulonephritis with hypernitremia by administration of anti-glomerular basement membrane (GBM) antibodies, all FcR ␥ -deficient mice survived. Histologically, wild-type mice showed glomerular hypercellularity and thrombotic changes, whereas the renal tissue in FcR ␥ -deficient mice was almost intact. Deposition of anti-GBM antibody as well as complement components in the GBM were equally observed in both wild-type and knockout mice. These results demonstrate that the triggering of this type of glomerulonephritis is completely dependent on

The P2X7 receptor mediates extracellular ATP signaling implicated in the development of devastating diseases such as chronic pain and cancer. Activation of the P2X7 receptor leads to opening of the characteristic dye-permeable membrane pore for molecules up to ~900 Da. However, it remains controversial what constitutes this peculiar pore and how it opens. Here we show that the panda receptor, when purified and reconstituted into liposomes, forms an intrinsic dye-permeable pore in the absence of other cellular components. Unexpectedly, we found that this pore opens independent of its unique C-terminal domain. We also found that P2X7 channel activity is facilitated by phosphatidylglycerol and sphingomyelin, but dominantly inhibited by cholesterol through direct interactions with the transmembrane domain. In combination with cell-based functional studies, our data suggest that the P2X7 receptor itself constitutes a lipid-composition dependent dye-permeable pore, whose opening is facilitated by palmitoylated cysteines near the pore-lining helix.

Diuretic activities of xanthine or nonxanthine adenosine antagonists and their ameliorative effects against glycerol-induced acute renal failure in rats were investigated in order to clarify the physiological and pathological function of adenosine receptors in the kidney. Diuretic and natriuretic activities of a variety of adenosine antagonists clarified systematically for the first time that the blockade of A1 receptors is more important than that of A2 receptors in sodium and water excretion and support the hypothesis that endogenous intrarenal levels of adenosine directly enhance tubular sodium readsorption. Studies of structure-activity relationships of 8-substituted xanthines in the acute renal failure demonstrated that the activation of adenosine A1 receptor was an important factor in developing such a renal failure. A series of 8-(3-noradamantyl)xanthines exhibited the extremely potent diuretic and natriuretic activities (24; 2.5 micrograms/kg, po, the ratio of urinary excretion value in treated rats to urinary excretion value in control rats = 1.69, the ratio of Na+/K+ in treated rats to Na+/K+ in control rats = 1.76) and potent ameliorative effects against glycerol-induced acute renal failure (24; 10 micrograms/kg, ip, 55% inhibition). From our detailed studies of structure-activity relationships, we can speculate that some tissue differences of the adenosine A1 receptor might exist between kidney and brain and sites of action for adenosine antagonists could be different between two renal pharmacological assays. 1,3-Dipropyl-8-(3-noradamantyl)xanthine, KW-3902 (24), was chosen for further studies and is under development as a drug for treating the acute renal failure.

Adenosine is a purine nucleoside and modulates a variety of physiological functions by interacting with cell-surface adenosine receptors. Under several adverse conditions, including ischemia, trauma, stress, seizures and inflammation, extracellular levels of adenosine are increased due to increased energy demands and ATP metabolism. Increased adenosine could protect against excessive cellular damage and organ dysfunction. Indeed, several protective effects of adenosine have been widely reported (e.g., amelioration of ischemic heart and brain injury, seizures and inflammation). However, the effects of adenosine itself are insufficient because extracellular adenosine is rapidly taken up into adjacent cells and subsequently metabolized. Adenosine uptake inhibitors (nucleoside transport inhibitors) could retard the disappearance of adenosine from the extracellular space by blocking adenosine uptake into cells. Therefore, it is expected that adenosine uptake inhibitors will have protective effects in various diseases, by elevating extracellular adenosine levels. Protective or ameliorating effects of adenosine uptake inhibitors in ischemic cardiac and cerebral injury, organ transplantation, seizures, thrombosis, insomnia, pain, and inflammatory diseases have been reported. Preclinical and clinical results indicate the possibility of therapeutic application of adenosine uptake inhibitors.

BackgroundThe ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.Principal FindingsThe binding of glycine betaine to purified OpuA and OpuAC (KD = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.ConclusionsIonic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.

KW-6002, a xanthine-based adenosine A(2A) antagonist, was labelled with the positron emitter carbon-11 by O-methylation of its precursor, KF23325, using [(11)C]iodomethane and was evaluated in rats as a putative in vivo radioligand for positron emission tomography (PET). Following intravenous injection of [(11)C]KW-6002, radioactivity was measured in blood, plasma, peripheral tissues, and in discrete brain tissues over a 2-h time period commensurate with PET scanning. In brain, [(11)C]KW-6002 showed highest retention in striata, with evidence of saturable binding, and lowest retention in frontal cortex (a tissue low in adenosine A(2A) receptors). PET scanning with [(11)C]KW-6002 demonstrated a specific signal in the striata which could be described using compartmental modelling. Specific binding was, however, also detected in extrastriatal regions, including brain areas reported to have low adenosine A(2A) receptor density. Blocking studies with the A(1) selective antagonist KF15372 and the non xanthine-type A(2A) antagonist ZM 241385 failed to elucidate the nature of this binding. Thus, although [(11)C]KW-6002 shows some potential for development as a PET ligand for quantifying striatal adenosine A(2A) receptor function, its in vivo selectivity requires further investigation.

Background: ABC transporter OpuA controls cell volume. Results: Anionic lipids are required for gating and efficient energy coupling of transport. Conclusion: Ionic strength and excluded volume effects act synergistically in the gating of transport. Significance: Tight coupling between substrate binding and ATP hydrolysis in ABC transporters allows the study of transmembrane signaling in nanodiscs.