Silvia Penuela

Pannexin 1 channels mediate /`find-me/' signal release and membrane permeability during apoptosis

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins

Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional intercellular communication. Localization of exogenously expressed pannexin 1 (Panx1) and pannexin 3 (Panx3), together with pharmacological studies, revealed a cell surface distribution profile and life cycle dynamics that were distinct from connexin 43 (Cx43, encoded by Gja1). Furthermore, N-glycosidase treatment showed that both Panx1 (∼41-48 kD species) and Panx3 (∼43 kD) were glycosylated, whereas N-linked glycosylation-defective mutants exhibited a decreased ability to be transported to the cell surface. Tissue surveys revealed the expression of Panx1 in several murine tissues – including in cartilage, skin, spleen and brain – whereas Panx3 expression was prevalent in skin and cartilage with a second higher-molecular-weight species present in a broad range of tissues. Tissue-specific localization patterns of Panx1 and Panx3 ranging from distinct cell surface clusters to intracellular profiles were revealed by immunostaining of skin and spleen sections. Finally, functional assays in cultured cells transiently expressing Panx1 and Panx3 were incapable of forming intercellular channels, but assembled into functional cell surface channels. Collectively, these studies show that Panx1 and Panx3 have many characteristics that are distinct from Cx43 and that these proteins probably play an important biological role as single membrane channels.

Pannexin channels are not gap junction hemichannels

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 channels 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.

Pannexins in ischemia-induced neurodegeneration

Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) form large-pore nonselective channels in the plasma membrane of cells and were suggested to play a role in the pathophysiology of cerebral ischemia. To directly test a potential contribution of pannexins in ischemia-related mechanisms, we performed experiments in Px1−/−, Px2−/−, and Px1−/−Px2−/− knockout mice. IL-1β release, channel function in astrocytes, and cortical spreading depolarization were not altered in Px1−/−Px2−/− mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons was lower, indicating that channel function in Px1−/−Px2−/− neurons was impaired. Furthermore, Px1−/−Px2−/− mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. In conclusion, our data demonstrate that Px1 and Px2 underlie channel function in neurons and contribute to ischemic brain damage.

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.

Glycosylation Regulates Pannexin Intermixing and Cellular Localization

The pannexin family of mammalian proteins, composed of Panx1, Panx2, and Panx3, has been postulated to be a new class of single-membrane channels with functional similarities to connexin gap junction proteins. In this study, immunolabeling and coimmunoprecipitation assays revealed that Panx1 can interact with Panx2 and to a lesser extent, with Panx3 in a glycosylation-dependent manner. Panx2 strongly interacts with the core and high-mannose species of Panx1 but not with Panx3. Biotinylation and dye uptake assays indicated that all three pannexins, as well as the N-glycosylation-defective mutants of Panx1 and Panx3, can traffic to the cell surface and form functional single-membrane channels. Interestingly, Panx2, which is also a glycoprotein and seems to only be glycosylated to a high-mannose form, is more abundant in intracellular compartments, except when coexpressed with Panx1, when its cell surface distribution increases by twofold. Functional assays indicated that the combination of Panx1 and Panx2 results in compromised channel function, whereas coexpressing Panx1 and Panx3 does not affect the incidence of dye uptake in 293T cells. Collectively, these results reveal that the functional state and cellular distribution of mouse pannexins are regulated by their glycosylation status and interactions among pannexin family members.

Pannexin1 Regulates α1-Adrenergic Receptor– Mediated Vasoconstriction

Rationale: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction. Objective: We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries. Methods and Results: Western blot, immunohistochemistry, and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and 10Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and &agr;1D-adrenergic receptor. Conclusions: Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.

Implications of pannexin 1 and pannexin 3 for keratinocyte differentiation

Pannexin (Panx) 1 and Panx3 are integral membrane proteins that share some sequence homology with the innexin family of invertebrate gap junctions. They are expressed in mammalian skin. Pannexins have been reported to form functional mechanosensitive single-membrane channels, but their importance in regulating cellular function is poorly understood. In this study, Panx1 and Panx3 were detected in the epidermis of 13.5 day embryonic mice. Compared with newborn mice, there was less Panx1 expression in both thin and thick murine skin, whereas Panx3 expression was unchanged. To investigate the role of pannexins in keratinocyte differentiation, we employed rat epidermal keratinocytes (REKs) that have the capacity to differentiate into organotypic epidermis, and engineered them to overexpress Panx1, Panx1-GFP or Panx3. The expression of Panx1 or Panx3 resulted in the increased ability of REKs to take up dye, suggesting that cell-surface channels were formed. Compared with monolayer REKs, endogenous Panx1 levels remained unchanged, whereas the 70 kDa immunoreactive species of Panx3 was greatly increased in the organotypic epidermis. In monolayer cultures, ectopic Panx1 and Panx1-GFP localized to the plasma membrane, whereas Panx3 displayed both intracellular and plasma-membrane profiles. Although both pannexins reduced cell proliferation, only Panx1 disrupted the architecture of the organotypic epidermis and markedly dysregulated cytokeratin 14 expression and localization. Furthermore, ectopic expression of only Panx1 reduced the vital layer thickness of the organotypic epidermis. In summary, Panx1 and Panx3 are coexpressed in the mammalian epidermis, and the regulation of Panx1 plays a key role in keratinocyte differentiation.

Pannexin channels and their links to human disease

In less than a decade, a small family of channel-forming glycoproteins, named pannexins, have captured the interest of many biologists, in large part due to their association with common diseases, ranging from cancers to neuropathies to infectious diseases. Although the pannexin family consists of only three members (Panx1, Panx2 and Panx3), one or more of these pannexins are expressed in virtually every mammalian organ, implicating their potential role in a diverse array of pathophysiologies. Panx1 is the most extensively studied, but features of this pannexin must be cautiously extrapolated to the other pannexins, as for example we now know that Panx2, unlike Panx1, exhibits unique properties such as a tendency to be retained within intracellular compartments. In the present review, we assess the biochemical and channel features of pannexins focusing on the literature which links these unique molecules to over a dozen diseases and syndromes. Although no germ-line mutations in genes encoding pannexins have been linked to any diseases, many cases have shown that high pannexin expression is associated with disease onset and/or progression. Disease may also occur, however, when pannexins are underexpressed, highlighting that pannexin expression must be exquisitely regulated. Finally, we discuss some of the most pressing questions and controversies in the pannexin field as the community seeks to uncover the full biological relevance of pannexins in healthy organs and during disease.

Pannexin1 and Pannexin3 Delivery, Cell Surface Dynamics, and Cytoskeletal Interactions

Pannexins (Panx) are a class of integral membrane proteins that have been proposed to exhibit characteristics similar to those of connexin family members. In this study, we utilized Cx43-positive BICR-M1Rk cells to stably express Panx1, Panx3, or Panx1-green fluorescent protein (GFP) to assess their trafficking, cell surface dynamics, and interplay with the cytoskeletal network. Expression of a Sar1 dominant negative mutant revealed that endoplasmic reticulum to Golgi transport of Panx1 and Panx3 was mediated via COPII-dependent vesicles. Distinct from Cx43-GFP, fluorescence recovery after photobleaching studies revealed that both Panx1-GFP and Panx3-GFP remained highly mobile at the cell surface. Unlike Cx43, Panx1-GFP exhibited no detectable interrelationship with microtubules. Conversely, cytochalasin B-induced disruption of microfilaments caused a severe loss of cell surface Panx1-GFP, a reduction in the recoverable fraction of Panx1-GFP that remained at the cell surface, and a decrease in Panx1-GFP vesicular transport. Furthermore, co-immunoprecipitation and co-sedimentation assays revealed actin as a novel binding partner of Panx1. Collectively, we conclude that although Panx1 and Panx3 share a common endoplasmic reticulum to Golgi secretory pathway to Cx43, their ultimate cell surface residency appears to be independent of cell contacts and the need for intact microtubules. Importantly, Panx1 has an interaction with actin microfilaments that regulates its cell surface localization and mobility.