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IntroductionP2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems.MethodsHere, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis.ResultsNo detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4-/- mice.DiscussionIn summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Abstract Background Over recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer’s disease (AD), and the ubiquitin–proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction. Methods To evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R. Results We describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of β5 and β1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival. Conclusions Our work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.

Abstract Background Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. Methods Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1β release after incubation with the P2X7-specific nbs. Results Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1β release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood–brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. Conclusion Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.

The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing “cap” domain, and a globular “ballast domain” containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)–2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.

The P2X7 ion channel is a key sensor for extracellular ATP and a key trigger of sterile inflammation. Intravenous injection of nanobodies that block P2X7 has shown to be beneficial in mouse models of systemic inflammation. P2X7 has also emerged as an attractive therapeutic target for inflammatory brain diseases. However, little is known about the ability of nanobodies to cross the BBB. Here we evaluated the ability of P2X7-specific nanobodies to reach and to block P2X7 on microglia following intravenous or intracerebral administration. For this study, we reformatted and sequence-optimized P2X7 nanobodies for higher stability and elevated isoelectric point. Following injection of nanobodies or nanobody-encoding adeno-associated viral vectors (AAV), we monitored the occupancy and blockade of microglial P2X7 in vivo using ex vivo flow cytometry. Our results show that P2X7 on microglia was within minutes completely occupied and blocked by intracerebroventricularly injected nanobodies, even at low doses. In contrast, very high doses were required to achieve similar effects when injected intravenously. The endogenous production of P2X7-antagonistic nanobodies following intracerebral or intramuscular injection of nanobody-encoding AAVs resulted in a long-term occupancy and blockade of P2X7 on microglia. Our results provide new insights into the conditions for the delivery of nanobodies to microglial P2X7 and point to AAV-mediated delivery of P2X7 nanobodies as a promising strategy for the treatment of sterile brain inflammation.

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

Summary: Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several transgenic mice carry a P2rx7 passenger mutation that affects the function of T cells. By the example of P2rx4tm1Rass we demonstrate that P2X4ko T cells express higher levels of P2X7 and are more sensitive toward the P2X7 activators ATP and NAD+, rendering these cells more vulnerable toward NAD-induced cell death (NICD) compared with wild type (WT). The enhanced NICD sensitivity confounded functional assays e.g. cytokine production and cell migration. Our results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes.

Background: It has been consistently reported that the deficiency of the adenosine triphosphate (ATP) sensitive purinergic receptor P2X7 (P2X7R) ameliorates symptoms in animal models of brain diseases.Objective: This study aimed to investigate the role of P2X7R in rodent models of acute and subchronic schizophrenia based on phencyclidine (PCP) delivery in animals lacking or overexpressing P2X7R, and to identify the underlying mechanisms involved.Methods: The psychotomimetic effects of acute i.p. PCP administration in C57Bl/6J wild-type, P2X7R knockout (P2rx7−/−) and overexpressing (P2X7-EGFP) young adult mice were quantified. The medial prefrontal cortex (mPFC) of P2rx7−/− and heterozygous P2X7-EGFP acutely treated animals was characterized through immunohistochemical staining. The prefrontal cortices of young adult P2rx7−/− and P2rx7tg/+ mice were examined with tritiated dopamine release experiments and the functional properties of the mPFC pyramidal neurons in layer V from P2rx7−/− mice were assessed by patch-clamp recordings. P2rx7−/− animals were subjected to a 7 days subchronic systemic PCP treatment. The animals working memory performance and PFC cytokine levels were assessed.Results: Our data strengthen the hypothesis that P2X7R modulates schizophrenia-like positive and cognitive symptoms in NMDA receptor antagonist models in a receptor expression level-dependent manner. P2X7R expression leads to higher medial PFC susceptibility to PCP-induced circuit hyperactivity. The mPFC of P2X7R knockout animals displayed distinct alterations in the neuronal activation pattern, microglial organization, specifically around hyperactive neurons, and were associated with lower intrinsic excitability of mPFC neurons.Conclusions: P2X7R expression exacerbated PCP-related effects in C57Bl/6J mice. Our findings suggest a pleiotropic role of P2X7R in the mPFC, consistent with the observed behavioral phenotype, regulating basal dopamine concentration, layer-specific neuronal activation, intrinsic excitability of neurons in the mPFC, and the interaction of microglia with hyperactive neurons. Direct measurements of P2X7R activity concerning microglial ramifications and dynamics could help to further elucidate the molecular mechanisms involved.

The ionotropic ATP-gated P2X7 receptor is an important contributor to inflammatory signaling cascades via the release of Interleukin-1β, as well as having roles in cell death, neuronal plasticity and the release of neurotransmitters. Accordingly, there is interest in targeting the P2X7 receptor for the treatment of epilepsy. However, the signaling pathways downstream of P2X7 receptor activation remain incompletely understood. Notably, recent studies showed that P2X7 receptor expression is controlled, in part, by microRNAs (miRNAs). Here, we explored P2X7 receptor-dependent microRNA expression by comparing microRNA expression profiles of wild-type (wt) and P2X7 receptor knockout mice before and after status epilepticus. Genome-wide microRNA profiling was performed using hippocampi from wt and P2X7 receptor knockout mice following status epilepticus induced by intra-amygdala kainic acid. This revealed that the genetic deletion of the P2X7 receptor results in distinct patterns of microRNA expression. Specifically, we found that in vehicle-injected control mice, the lack of the P2X7 receptor resulted in the up-regulation of 50 microRNAs and down-regulation of 35 microRNAs. Post-status epilepticus, P2X7 receptor deficiency led to the up-regulation of 44 microRNAs while 13 microRNAs were down-regulated. Moreover, there was only limited overlap among identified P2X7 receptor-dependent microRNAs between control conditions and post-status epilepticus, suggesting that the P2X7 receptor regulates the expression of different microRNAs during normal physiology and pathology. Bioinformatic analysis revealed that genes targeted by P2X7 receptor-dependent microRNAs were particularly overrepresented in pathways involved in intracellular signaling, inflammation, and cell death; processes that have been repeatedly associated with P2X7 receptor activation. Moreover, whereas genes involved in signaling pathways and inflammation were common among up- and down-regulated P2X7 receptor-dependent microRNAs during physiological and pathological conditions, genes associated with cell death seemed to be restricted to up-regulated microRNAs during both physiological conditions and post-status epilepticus. Taken together, our results demonstrate that the P2X7 receptor impacts on the expression profile of microRNAs in the brain, thereby possibly contributing to both the maintenance of normal cellular homeostasis and pathological processes.

Circulating molecules have potential as biomarkers to support the diagnosis of epilepsy and to assist with differential diagnosis, for example, in conditions resembling epilepsy, such as in psychogenic non-epileptic seizures (PNES). The P2X7 receptor (P2X7R) is an important regulator of inflammation and mounting evidence supports its activation in the brain during epilepsy. Whether the P2X7R or P2X7R-dependent signaling molecules can be used as biomarkers of epilepsy has not been reported. P2X7R levels were analyzed by quantitative ELISA using plasma samples from controls and patients with temporal lobe epilepsy (TLE) or PNES. Moreover, blood cell P2X7R expression and P2X7R-dependent cytokine signature was measured following status epilepticus in P2X7R-EGFP reporter, wildtype, and P2X7R-knockout mice. P2X7R plasma levels were higher in TLE patients when compared with controls and patients with PNES. Plasma levels of the broad inflammatory marker protein C-Reactive protein (CRP) were similar between the three groups. Using P2X7R-EGFP reporter mice, we identified monocytes as the main blood cell type expressing P2X7R after experimentally evoked seizures. Finally, cytokine array analysis in P2X7R-deficient mice identified KC/GRO as a potential P2X7R-dependent plasma biomarker following status epilepticus and during epilepsy. Our data suggest that P2X7R signaling components may be a promising subclass of circulating biomarkers to support the diagnosis of epilepsy.

Extracellular adenine nucleotides play important roles in cell–cell communication and tissue homeostasis. High concentrations of extracellular ATP released by dying cells are sensed as a danger signal by the P2X7 receptor, a non-specific cation channel. Studies in P2X7 knockout mice and numerous disease models have demonstrated an important role of this receptor in inflammatory processes. P2X7 activation has been shown to induce a variety of cellular responses that are not usually associated with ion channel function, for example changes in the plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis. In contrast to all other P2X family members, the P2X7 receptor contains a long intracellular C-terminus that constitutes 40% of the whole protein and is considered essential for most of these effects. So far, over 50 different proteins have been identified to physically interact with the P2X7 receptor. However, few of these interactions have been confirmed in independent studies and for the majority of these proteins, the interaction domains and the physiological consequences of the interactions are only poorly described. Also, while the structure of the P2X7 extracellular domain has recently been resolved, information about the organization and structure of its C-terminal tail remains elusive. After shortly describing the structure and assembly of the P2X7 receptor, this review gives an update of the identified or proposed interaction domains within the P2X7 C-terminus, describes signaling pathways in which this receptor has been involved, and provides an overlook of the identified interaction partners.

Peptides derived from animal venoms provide important research tools for biochemical and pharmacological characterization of receptors, ion channels, and transporters. Some venom peptides have been developed into drugs (such as the synthetic ω-conotoxin MVIIA, ziconotide) and several are currently undergoing clinical trials for various clinical indications. Challenges in the development of peptides include their usually limited supply from natural sources, cost-intensive chemical synthesis, and potentially complicated stereoselective disulfide-bond formation in the case of disulfide-rich peptides. In particular, if extended structure–function analysis is performed or incorporation of stable isotopes for NMR studies is required, the comparatively low yields and high costs of synthesized peptides might constitute a limiting factor. Here we investigated the expression of the 4/7 α-conotoxin TxIA, a potent blocker at α3β2 and α7 nicotinic acetylcholine receptors (nAChRs), and three analogs in the form of maltose binding protein fusion proteins in Escherichia coli. Upon purification via nickel affinity chromatography and release of the toxins by protease cleavage, HPLC analysis revealed one major peak with the correct mass for all peptides. The final yield was 1–2 mg of recombinant peptide per liter of bacterial culture. Two-electrode voltage clamp analysis on oocyte-expressed nAChR subtypes demonstrated the functionality of these peptides but also revealed a 30 to 100-fold potency decrease of expressed TxIA compared to chemically synthesized TxIA. NMR spectroscopy analysis of TxIA and two of its analogs confirmed that the decreased activity was due to an alternative disulfide linkage rather than the missing C-terminal amidation, a post-translational modification that is common in α-conotoxins. All peptides preferentially formed in the ribbon conformation rather than the native globular conformation. Interestingly, in the case of the α7 nAChR, but not the α3β2 subtype, the loss of potency could be rescued by an R5D substitution. In conclusion, we demonstrate efficient expression of functional but alternatively folded ribbon TxIA variants in E. coli and provide the first structure–function analysis for a ribbon 4/7-α-conotoxin at α7 and α3β2 nAChRs. Computational analysis based on these data provide evidence for a ribbon α-conotoxin binding mode that might be exploited to design ligands with optimized selectivity.

Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3β2 and α6/α3β2β3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.

Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins.

The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.

Venoms from cone snails (Conidae) have been extensively studied during the last decades, but those from other members of the suborder Toxoglossa, such as of Terebridae and Turridae superfamilies attracted less interest so far. Here, we report the effects of venom and gland extracts from three species of the superfamily Terebridae. By 2-electrode voltage-clamp technique the gland extracts were tested on Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) of rat neuronal (α3β2, α3β4, α4β2, α4β4, α7) and muscle subtypes (α1β1γδ), and expressing potassium (Kv1.2 and Kv1.3) and sodium channels (Nav1.2, 1.3, 1.4, 1.6). The extracts were shown to exhibit remarkably high inhibitory activities on almost all nAChRs tested, in particular on the α7 subtype suggesting the presence of peptides of the A-superfamily from the venom of Conus species. In contrast, no effects on the potassium and sodium channels tested were observed. The venoms of terebrid snails may offer an additional source of novel biologically active peptides.

Cone snails produce a fast-acting and often paralyzing venom that is usually injected into their prey or predator through a hypodermic needle-like modified radula tooth. Many diverse compounds are found in their venom including small molecules, peptides and enzymes. However, peptidic toxins called conotoxins (10–40 residues and 2–4 disulfide bonds) largely dominate these cocktails. These disulfide rich toxins are very valuable pharmacological tools for investigating the function of ions channels, G-protein coupled receptors, transporters and enzymes. Here, we report on the synthesis, structure determination and biological activities of two α-conotoxins, CIA and CIB, found in the predatory venom of the piscivorous species Conus catus. CIA is a typical 3/5 α-conotoxin that blocks the rat muscle type nAChR with an IC50 of 5.7 nM. Interestingly, CIA also inhibits the neuronal rat nAChR subtype α3β2 with an IC50 of 2.06 μM. CIB is a 4/7 α-conotoxin that blocks rat neuronal nAChR subtypes, including α3β2 (IC50 = 128.9 nM) and α7 (IC50 = 1.51 μM). High resolution NMR structures revealed typical α-conotoxin folds for both peptides. We also investigated the in vivo effects of these toxins on fish, since both peptides were identified in the predatory venom of C. catus. Consistent with their pharmacology, CIA was highly paralytic to zebrafish (ED50 = 110 μg/kg), whereas CIB did not affect the mobility of the fish. In conclusion, CIA likely participates in prey capture through muscle paralysis, while the putative ecological role of CIB remains to be elucidated.

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.