Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

Pannexins constitute a family of three glycoproteins (Panx1, -2, and -3) forming single membrane channels. Recent work demonstrated that Panx1 is expressed in skeletal muscle and involved in the potentiation of contraction. However, Panxs functions in skeletal muscle cell differentiation, and proliferation had yet to be assessed. We show here that Panx1 and Panx3, but not Panx2, are present in human and rodent skeletal muscle, and their various species are differentially expressed in fetal versus adult human skeletal muscle tissue. Panx1 levels were very low in undifferentiated human primary skeletal muscle cells and myoblasts (HSMM) but increased drastically during differentiation and became the main Panx expressed in differentiated cells. Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone. As for Panx3, its lower molecular weight species were prominent in adult skeletal muscle but very low in the fetal tissue and in undifferentiated skeletal muscle cells and myoblasts. Its overexpression (∼43-kDa species) induced HSMM differentiation and also inhibited their proliferation. On the other hand, a ∼70-kDa immunoreactive species of Panx3, likely glycosylated, sialylated, and phosphorylated, was highly expressed in proliferative myoblasts but strikingly down-regulated during their differentiation. Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation. In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.     

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.     

Pannexin 3 Regulates Intracellular ATP/cAMP Levels and Promotes Chondrocyte Differentiation

Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.     

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.     

Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation

The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca(2+) channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca(2+) waves between cells. Blocking the Panx3 Ca(2+) channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca(2+) channel and a hemichannel, and by forming gap junctions.     

Pannexin 2 is expressed in murine skin and promotes UVB-induced apoptosis of keratinocytes

Pannexins (PANX) are a family of three channel-forming membrane glycoproteins expressed in the skin. Previous studies have focused on the role of PANX1 and PANX3 in the regulation of cellular functions in skin cells while PANX2, the largest member of this protein family, has not been investigated. In the current study, we explored the temporal PANX2 expression in murine skin and found that one Panx2 splice variant (Panx2-202) tends to be more abundant at the protein level and is continuously expressed in developed skin. PANX2 was detected in the suprabasal layers of the mouse epidermis and up-regulated in an in vitro model of rat epidermal keratinocyte differentiation. Furthermore, we show that in apoptotic rat keratinocytes, upon UV light B (UVB)-induced caspase-3/7 activation, ectopically overexpressed PANX2 is cleaved in its C-terminal domain at the D416 residue without increasing the apoptotic rate measured by caspase-3/7 activation. Notably, CRISPR-Cas9 mediated genetic deletion of rat Panx2 delays but does not impair caspase-3/7 activation and cytotoxicity in UVB-irradiated keratinocytes. We propose that endogenous PANX2 expression in keratinocytes promotes cell death after UVB insult and may contribute to skin homeostasis.     

The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins

We have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant. The human and mouse pannexin-1 mRNAs are ubiquitously, although disproportionately, expressed in normal tissues. Human PANX2 is a brain-specific gene; its mouse orthologue, Panx2, is also expressed in certain cell types in developing brain. In silico evaluation of Panx3 expression predicts gene expression in osteoblasts and synovial fibroblasts. The apparent conservation of pannexins between species merits further investigation.     

Diverse subcellular distribution profiles of pannexin 1 and pannexin 3

Pannexins have been proposed to play a role in gap junctional intercellular communication and as single-membrane channels, although many of their molecular characteristics differ from connexins. Localization of untagged Panx1 and Panx3 exogenously expressed in five cultured cell lines revealed a cell surface distribution profile with limited evidence of cell surface clustering and variable levels of intracellular pools. However, N-glycosylation-defective mutants of pannexins exhibited a more prominent intracellular distribution with decreased cell surface labeling, suggesting an important role for pannexin glycosylation in trafficking. Similar to wild-type pannexins, the glycosylation-defective mutants failed to noticeably transfer microinjected fluorescent dyes to neighboring cells, suggesting that few, or no functional intercellular channels were formed. Finally, varied distribution patterns of endogenous Panx1 and Panx3 were observed in cells of osteoblast origin and Madin-Darby canine kidney cells. Collectively, diverse expression and distribution profiles of Panx1 and Panx3 suggest that they may have multiple cellular functions.     

Pannexin1 Channel Proteins in the Zebrafish Retina Have Shared and Unique Properties

In mammals, a single pannexin1 gene (Panx1) is widely expressed in the CNS including the inner and outer retinae, forming large-pore voltage-gated membrane channels, which are involved in calcium and ATP signaling. Previously, we discovered that zebrafish lack Panx1 expression in the inner retina, with drPanx1a exclusively expressed in horizontal cells of the outer retina. Here, we characterize a second drPanx1 protein, drPanx1b, generated by whole-genome duplications during teleost evolution. Homology searches strongly support the presence of pannexin sequences in cartilaginous fish and provide evidence that pannexins evolved when urochordata and chordata evolution split. Further, we confirm Panx1 ohnologs being solely present in teleosts. A hallmark of differential expression of drPanx1a and drPanx1b in various zebrafish brain areas is the non-overlapping protein localization of drPanx1a in the outer and drPanx1b in the inner fish retina. A functional comparison of the evolutionary distant fish and mouse Panx1s revealed both, preserved and unique properties. Preserved functions are the capability to form channels opening at resting potential, which are sensitive to known gap junction and hemichannel blockers, intracellular calcium, extracellular ATP and pH changes. However, drPanx1b is unique due to its highly complex glycosylation pattern and distinct electrophysiological gating kinetics. The existence of two Panx1 proteins in zebrafish displaying distinct tissue distribution, protein modification and electrophysiological properties, suggests that both proteins fulfill different functions in vivo.     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Interactions of Pannexin1 channels with purinergic and NMDA receptor channels

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

What is hidden in the pannexin treasure trove: the sneak peek and the guesswork

Connexins had been considered to be the only class of the vertebrate proteins capable of gap junction formation; however, new candidates for this function with no homology to connexins, termed pannexins were discovered. So far three pannexins were described in rodent and human genomes: Panx1, Panx2 and Panx3. Expressions of pannexins can be detected in numerous brain structures, and now found both in neuronal and glial cells. Hypothetical roles of pannexins in the nervous system include participating in sensory processing, hippocampal plasticity, synchronization between hippocampus and cortex, and propagation of the calcium waves supported by glial cells, which help maintain and modulate neuronal metabolism. Pannexin also may participate in pathological reactions of the neural cells, including their damage after ischemia and subsequent cell death. Recent study revealed non-gap junction function of Panx1 hemichannels in erythrocytes, where they serve as the conduits for the ATP release in response to the osmotic stress. High-throughput studies produced some evidences of the pannexin involvement in the process of tumorigenesis. According to brain cancer gene expression database REMBRANDT, PANX2 expression levels can predict post diagnosis survival for patients with glial tumors. Further investigations are needed to verify or reject hypotheses listed.     

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-M1R_k 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.     

Intrinsic properties and regulation of Pannexin 1 channel

Pannexin 1 (Panx1) channels are generally represented as non-selective, large-pore channels that release ATP. Emerging roles have been described for Panx1 in mediating purinergic signaling in the normal nervous, cardiovascular, and immune systems, where they may be activated by mechanical stress, ionotropic and metabotropic receptor signaling, and via proteolytic cleavage of the Panx1 C-terminus. Panx1 channels are widely expressed in various cell types, and it is now thought that targeting these channels therapeutically may be beneficial in a number of pathophysiological contexts, such as asthma, atherosclerosis, hypertension, and ischemic-induced seizures. Even as interest in Panx1 channels is burgeoning, some of their basic properties, mechanisms of modulation, and proposed functions remain controversial, with recent reports challenging some long-held views regarding Panx1 channels. In this brief review, we summarize some well-established features of Panx1 channels; we then address some current confounding issues surrounding Panx1 channels, especially with respect to intrinsic channel properties, in order to raise awareness of these unsettled issues for future research.     

Regulation of pannexin channels by post-translational modifications

The large-pore channels formed by the pannexin family of proteins have been implicated in many physiological and pathophysiological functions, mainly through their ATP release function. However, a tight regulation of channel opening is necessary to modulate their function in vivo. Post-translational modifications have been postulated as some of the regulating mechanisms for Panx1, while Panx2 and Panx3 have not been as well characterized. Positive regulators include caspase cleavage to open Panx1 channels in apoptotic cells, and activation by Src family kinases via ionotropic receptors in neurons and macrophages. S-nitrosylation of cysteines has been shown to both inhibit and activate the Panx1 channel in different cell types. All three pannexins are N-glycosylated but to different levels of modification. Their diverse glycosylation appears to regulate cellular localization, intermixing, and may restrict their ability to function as inter-cellular channels. It is clear that our understanding of pannexin post-translational modification and their role in channel function regulation is still in its infancy even a decade after their discovery.     

Contribution of Pannexin1 to Experimental Autoimmune Encephalomyelitis

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.     

Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.     

Connexins and pannexins in neuronal development and adult neurogenesis

Connexins and pannexins share very similar structures and functions; they also exhibit overlapping expression in many stages of neuronal development. Here, we review evidence implicating connexin- and pannexin-mediated communication in the regulation of the birth and development of neurons, specifically Cx26, Cx30, Cx32, Cx36, Cx43, Cx45, Panx1, and Panx2. We begin by dissecting the involvement of these proteins in the generation and development of new neurons in the embryonic, postnatal, and adult brain. Next we briefly outline common mechanisms employed by both pannexins and connexins in these roles, including modulation of purinergic receptor signalling and signalling nexus functions. Throughout this review we highlight developing themes as well as important gaps in knowledge to be bridged.