Deacetylation as a receptor-regulated direct activation switch for pannexin channels.
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ABSTRACT:
SUBMITTER: Chiu YH
PROVIDER: S-EPMC8302610 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Anoxic depolarization of pyramidal neurons results from a large inward current that is activated, in part, by excessive glutamate release during exposure to anoxia/ischemia. Pannexin-1 (Panx1) channels can be activated both by ischemia and NMDA receptors (NMDARs), but the mechanisms of Panx1 activation are unknown. We used whole-cell recordings to show that pharmacological inhibition or conditional genetic deletion of Panx1 strongly attenuates the anoxic depolarization of CA1 pyramidal neurons in acute brain slices from rats and mice. Anoxia or exogenous NMDA activated Src family kinases (SFKs), as measured by increased phosphorylation of SFKs at Y416. The SFK inhibitor PP2 prevented Src activation and Panx1 opening during anoxia. A newly developed interfering peptide that targets the SFK consensus-like sequence of Panx1 (Y308) attenuated the anoxic depolarization (AD) without affecting SFK activation. Importantly, the NMDAR antagonists, D-APV and R-CPP, attenuated AD currents carried by Panx1, and the combined application of D-APV and (10)panx (a Panx1 blocker) inhibited AD currents to the same extent as either blocker alone. We conclude that activation of NMDARs during anoxia/ischemia recruits SFKs to open Panx1, leading to sustained neuronal depolarizations.
| S-EPMC6621249 | biostudies-literature
Project description:Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.
| S-EPMC5290276 | biostudies-literature
Project description:Apoptosis can promote inflammation by triggering activation of the NLRP3 inflammasome (NLR family, pyrin domain containing 3). However, the molecular mechanisms regulating these processes are ill-defined. We recently reported that pannexin-1 is required to promote NLRP3 inflammasome assembly. We further demonstrate that differential cleavage of gasdermin D (GSDMD) by apoptotic caspases regulates inflammatory cell lysis. Here, we discuss our findings and perspectives for future studies.
| S-EPMC6548479 | biostudies-literature
Project description:BackgroundPannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ~1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.ObjectiveTo determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.MethodsPanx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca(2+) influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL(-1) apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.ResultsPANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca(2+) influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.ConclusionsPanx1 is expressed on human platelets and amplifies Ca(2+) influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.
| S-EPMC4238786 | biostudies-literature
Project description:ObjectiveBrown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of ?-adrenergic receptor (?AR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis.MethodsIn an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe.ResultsIn brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by ?3AR stimulation via a novel mechanism that involves G?? subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of ?3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective ?3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance.ConclusionsThese data demonstrate that G??-dependent Panx1 channel activation is involved in ?3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of ?3AR activation may have therapeutic implications.
| S-EPMC7779784 | biostudies-literature
Project description:Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity-measured by changes in DAPI uptake-of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water-lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.
| S-EPMC7731223 | biostudies-literature
Project description:Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mμn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mμn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. © 2020 American Society for Bone and Mineral Research.
| S-EPMC8009310 | biostudies-literature
Project description:Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers, or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term "hemichannels" to describe pannexin oligomers while others use the term "channels" instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus have an assembly and functional state as gap junctional intercellular channels. Here we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channel in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature.
| S-EPMC3704572 | biostudies-literature
Project description:Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1β and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.
| S-EPMC8938517 | biostudies-literature
Project description:Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. Here we show that the quinolone antibiotic trovafloxacin is a novel PANX1 inhibitor, by using a small-molecule screen. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials.
| S-EPMC4078991 | biostudies-literature