Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

The long ß2,3-sheets encoded by redundant sequences play an integral role in the channel function of P2X7 receptors.

P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened ß2- and ß3-sheets and their linker (loop ß2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer ß2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer ß2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype.

Tyrosine 288 in the extracellular domain of the human P2X7 receptor is critical for receptor function revealed by structural modeling and site-directed mutagenesis.

The P2X7 receptor (P2X7R) is a calcium-permeable cation channel activated by high concentrations of extracellular ATP. It plays a role in vital physiological processes, particularly in innate immunity, and is dysregulated in pathological conditions such as inflammatory diseases, neurodegenerative diseases, mood disorders, and cancers. Structural modeling of the human P2X7R (hP2X7R) based on the recently available structures of the rat P2X7 receptor (rP2XR) in conjunction with molecular docking predicts the orientation of tyrosine at position 288 (Y288) in the extracellular domain to face ATP. In this short communication, we combined site-directed mutagenesis and whole-cell patch-clamp recording to investigate the role of this residue in the hP2X7R function. Mutation of this extracellular residue to amino acids with different properties massively impaired current responses to both ATP and BzATP, suggesting that Y288 is important for normal receptor function. Such a finding facilitates development of an in-depth understanding of the molecular basis of hP2X7R structure-function relationships.

Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside-compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. SIGNIFICANCE STATEMENT: Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.

Dissecting activation steps in P2X7 receptors.

P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.

P2X7 receptor antagonist BBG inhibits endoplasmic reticulum stress and pyroptosis to alleviate postherpetic neuralgia.

Postherpetic neuralgia (PHN) is the most common complication of acute herpes zoster. The treatment of PHN remains a challenge for clinical pain management. The present study investigated the P2X7 receptor antagonist brilliant blue G (BBG) whether inhibits endoplasmic reticulum stress and pyroptosis (a necrotic form of cell death) and alleviates PHN. Varicella zoster virus (VZV)-infected CV-1 cells were used to induce PHN model. Mechanical paw withdrawal thresholds were measured using an ascending series of von Frey filaments. Immunohistochemistry was used to detect the expression of P2X7R in nerve tissues. Western blot was used to determine the expression of endoplasmic reticulum (ER) stress and pyroptosis-related molecules. The expression of IL-1ß and IL-18 in tissue homogenate was detected by ELISA. The PHN rat has the lower paw withdrawal threshold, but higher expression of P2X7 in nerve tissues. And, endoplasmic reticulum stress was activated and pyroptosis was increased in PHN rats. BBG can decrease pain thresholds and reduce ER stress and pyroptosis in PHN rats. In addition, ER stress activator tunicamycin (TM) can reverse the effect of BBG on the paw withdrawal thresholds, endoplasmic reticulum stress, and pyroptosis. Therefore, P2X7 receptor antagonist BBG alleviates PHN by activating ER stress and reducing pyroptosis.

Structural basis for the functional properties of the P2X7 receptor for extracellular ATP.

The P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure-function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.

P2X7 Receptors and TMEM16 Channels Are Functionally Coupled with Implications for Macropore Formation and Current Facilitation.

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.

Protective effects of P2X7R antagonist in sepsis-induced acute lung injury in mice via regulation of circ_0001679 and circ_0001212 and downstream Pln, Cdh2, and Nprl3 expression.

BACKGROUND: Sepsis induces pulmonary P2X7 receptor (P2X7 R) expression and P2X7 R-knockout reduced lung inflammation in mice. The present study investigated the expression of circular RNA (circRNA) and mRNA in sepsis-induced acute lung injury (ALI) treated with a P2X7 R antagonist. METHODS: Sepsis was induced by tracheal administration of lipopolysaccharide (LPS), and the mice were then divided into two groups: without [sepsis + dimethyl sulfoxide (DMSO)] or with P2X7 R antagonist treatment (sepsis + P2X7 A). Sham mice were administrated sterile normal saline. Serum levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, pathological changes, cell apoptosis and P2X7 R expression in lung were assessed, followed by RNA sequencing (RNA-seq) and bioinformatics analyses. A quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to validate circRNAs and mRNAs. RESULTS: Compared to the sham group, LPS-induced sepsis produced obvious pathological changes in lung tissue, as well as increased apoptotic lung cells, serum TNF-α and IL-1ß levels, and P2X7 R expression; P2X7 R antagonism significantly ameliorated these changes. RNA-seq identified many dysregulated circRNAs and mRNAs during sepsis, whereas this changed with P2X7 R antagonism. RT-qPCR confirmed that Mus musculus (mmu)_circ_0001679, mmu_circ_0001212, phospholamban (Pln), cadherin-2 (Cdh2) and nitrogen permease regulator 3-like (Nprl3) expression were significantly increased in the sepsis + DMSO group compared to that in the sham group but were decreased in the sepsis + P2X7 A group compared to that in the sepsis + DMSO group. The circRNA-microRNA-mRNA coexpression network indicated that mmu_circ_0001679 may regulate Nprl3 and that mmu_circ_0001212 may similarly regulate Pln, Cdh2 and Nprl3 as a competing endogenous RNA. CONCLUSIONS: P2X7 R antagonism attenuates sepsis-induced ALI by inhibiting dysregulated expression of circRNA (circ_0001679, circ_0001212) and mRNA (Pln, Cdh2 and Nprl3).

Production, characterization, and application of a monoclonal antibody specific for the extracellular domain of human P2X7R.

This paper focuses on the production of a high-affinity monoclonal antibody (mAb) that can efficiently detect and block purinergic ligand-gated ion channel 7 receptor (P2X7R). To achieve this goal, the extracellular domain of human P2X7R, P2X7R-ECD, was used as an immunogen for BALB/c mice, inducing them to produce spleen lymphocytes that were subsequently fused with myeloma cells. Screening of the resultant hybridoma clones resulted in the selection of one stable positive clone that produced a qualified mAb, named 4B3A4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purity of the purified 4B3A4 mAb was above 85%, with prominent bands corresponding to molecular weights of 55 kDa (heavy chain) and 25 kDa (light chain), and the BCA assay showed that the concentration of the purified 4B3A4 mAb was 0.3 mg/mL. Western blot analysis revealed that the 4B3A4 mAb could specifically recognize and bind both P2X7R-ECD and the full-length P2X7R protein. Laser scanning confocal microscopy (LSCM) revealed that the 4B3A4 mAb specifically bound to P2X7R on the membrane of human peripheral blood mononuclear cells (PBMCs). P2X7R expression was significantly different between healthy individuals and people with certain cancers as determined by flow cytometry (FCM). In addition, the 4B3A4 mAb significantly reduced ATP-stimulated Ca2+ entry and YO-PRO-1 uptake, which indicated that the 4B3A4 mAb effectively blocked P2X7R activity. These data indicate that the 4B3A4 mAb can be further used as not only an antibody to detect cell surface P2X7R but also as a therapeutic antibody to target P2X7R-related signaling pathways.

Localization of the gate and selectivity filter of the full-length P2X7 receptor.

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. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4.

Structural and Functional Basis for Understanding the Biological Significance of P2X7 Receptor.

The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels-inexistent in physiological conditions-are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling.

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

P2X7 Receptor at the Crossroads of T Cell Fate.

The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions.

Dihydrotanshinone, a Natural Diterpenoid, Preserves Blood-Retinal Barrier Integrity via P2X7 Receptor.

Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1ß, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.

Mapping the Site of Action of Human P2X7 Receptor Antagonists AZ11645373, Brilliant Blue G, KN-62, Calmidazolium, and ZINC58368839 to the Intersubunit Allosteric Pocket.

The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R-selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R-selective antagonists ZINC58368839, brilliant blue G, KN-62, and calmidazolium. The effect of intersubunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A, and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a binding mode similar to AZ11645373 and other previously characterized antagonists. For the larger antagonists, brilliant blue G, KN-62, and calmidazolium, our data imply an overlapping but distinct binding mode involving the central upper vestibule of the receptor in addition to the intersubunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists and establishes "signature mutants" for P2X7R binding-mode characterization.

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages.

Extracellular adenosine 5'-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1ß cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100 µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24 h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.

Mapping the Allosteric Action of Antagonists A740003 and A438079 Reveals a Role for the Left Flipper in Ligand Sensitivity at P2X7 Receptors.

P2X7 receptor (P2X7R) activation requires ∼100-fold higher concentrations of ATP than other P2X receptor (P2XR) subtypes. Such high levels are found during cellular stress, and P2X7Rs consequently contribute to a range of pathophysiological conditions. We have used chimeric and mutant P2X7Rs, coupled with molecular modeling, to produce a validated model of the binding mode of the subtype-selective antagonist A438079 at an intersubunit allosteric site. Within the allosteric site large effects on antagonist action were found for point mutants of residues F88A, D92A, F95A, and F103A that were conserved or similar between sensitive/insensitive P2XR subtypes, suggesting that these side-chain interactions were not solely responsible for high-affinity antagonist binding. Antagonist sensitivity was increased with mutations that remove the bulk of side chains around the center of the binding pocket, suggesting that the dimensions of the pocket make a significant contribution to selectivity. Chimeric receptors swapping the left flipper (around the orthosteric site) reduced both ATP and antagonist sensitivity. Point mutations within this region highlighted the contribution of a P2X7R-specific aspartic acid residue (D280) that modeling suggests forms a salt bridge with the lower body region of the receptor. The D280A mutant removing this charge increased ATP potency 15-fold providing a new insight into the low ATP sensitivity of the P2X7R. The ortho- and allosteric binding sites form either side of the ß-strand Y291-E301 adjacent to the left flipper. This structural linking may explain the contribution of the left flipper to both agonist and antagonist action.

Intrathecal delivery of a palmitoylated peptide targeting Y382-384 within the P2X7 receptor alleviates neuropathic pain.

Pain hypersensitivity resulting from peripheral nerve injury depends on pathological microglial activation in the dorsal horn of the spinal cord. This microglial activity is critically modulated by P2X7 receptors (P2X7R) and ATP stimulation of these receptors produces mechanical allodynia, a defining feature of neuropathic pain. Peripheral nerve injury increases P2X7R expression and potentiates its cation channel function in spinal microglia. Here, we report a means to preferentially block the potentiation of P2X7R function by delivering a membrane permeant small interfering peptide that targets Y382-384, a putative tyrosine phosphorylation site within the P2X7R intracellular C-terminal domain. Intrathecal administration of this palmitoylated peptide (P2X7R379-389) transiently reversed mechanical allodynia caused by peripheral nerve injury in both male and female rats. Furthermore, targeting Y382-384 suppressed P2X7R-mediated release of cytokine tumor necrosis factor alpha and blocked the adoptive transfer of mechanical allodynia caused by intrathecal injection of P2X7R-stimulated microglia. Thus, Y382-384 site-specific modulation of P2X7R is an important microglial mechanism in neuropathic pain.

Synthesis and preliminary biological evaluation of a novel P2X7R radioligand [18F]IUR-1601.

The reference standard IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from tert-butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoroethylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 12% in three steps. The target tracer [18F]IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-[18F]fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from desmethyl-GSK1482160 with 2-[18F]fluoroethyl tosylate, prepared from 1,2-ethylene glycol-bis-tosylate and K[18F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 1-3% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74-370 GBq/µmol. The potency of IUR-1601 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity Ki values for IUR-1601 and GSK1482160 are 4.31 and 5.14 nM, respectively.