Pannexins,distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ‐forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co‐expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.     

Innexins form two types of channels

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.     

Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts

The importance of connexins (Cxs) in cochlear functions has been demonstrated by the finding that mutations in Cx genes cause a large proportion of sensorineural hearing loss cases. However, it is still unclear how Cxs contribute to the cochlear function. Recent data (33) obtained from Cx30 knockout mice showing that a reduction of Cx diversity in assembling gap junctions is sufficient to cause deafness suggest that functional interactions of different subtypes of Cxs may be essential in normal hearing. In this work we show that the two major forms of Cxs (Cx26 and Cx30) in the cochlea have overlapping expression patterns beginning at early embryonic stages. Cx26 and Cx30 were colocalized in most gap junction plaques in the cochlea, and their coassembly was tested by coimmunoprecipitation. To compare functional differences of gap junctions with different molecular configurations, homo- and heteromeric gap junctions composed of Cx26 and/or Cx30 were reconstituted by transfections in human embryonic kidney-293 cells. The ratio imaging technique and fluorescent tracer diffusion assays were used to assess the function of reconstituted gap junctions. Our results revealed that gap junctions with different molecular configurations show differences in biochemical coupling, and that intercellular Ca²⁺ signaling across heteromeric gap junctions consisting of Cx26 and Cx30 was at least twice as fast as their homomerically assembled counterparts. Our data suggest that biochemical permeability and the dynamics of intercellular signaling through gap junction channels, in addition to gap junction-mediated intercellular ionic coupling, may be important factors to consider for studying functional roles of gap junctions in the cochlea. cochlea; coassembly; deafness     

Pannexin: to gap or not to gap, is that a question?

Vertebrates express two families of gap junction proteins: the well characterized connexins and the recently discovered pannexins. The latter are related to invertebrate innexins. Here we present the hypothesis that pannexins, rather than providing a redundant system to gap junctions formed by connexins, exert a physiological role as nonjunctional membrane channels. Specifically, we propose that pannexins can serve as ATP release channels. This function presumptively is also performed by innexins in invertebrates, in addition to their traditional gap junction role.     

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.     

Gap junction gene and protein families: Connexins,innexins, and pannexins

Gap junction channels facilitate the intercellular exchange of ions and small molecules. While this process is critical to all multicellular organisms, the proteins that form gap junction channels are not conserved. Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. Interestingly, vertebrates and lower chordates contain innexin homologs, the pannexins, which also form channels, but rarely (if ever) make intercellular channels. While the connexin and the innexin/pannexin polypeptides do not share significant sequence similarity, all three of these protein families share a similar membrane topology and some similarities in quaternary structure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

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 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.     

Intercellular channels in animals

Gap junctions are considered to serve a similar function in all multicellular animals (Metazoa). Two unrelated protein families are involved in this function: connexins, which are found only in chordates, and pannexins, which are present in the genomes of both chordates and invertebrates. Recent sequence data from different organisms show important exceptions to this simplified scheme. It looks as if Chordate lancelet has only pannexins and no connexins in its genome. New data indicate that some metazoans have neither connexins nor pannexins and use other unidentified proteins to form gap junctions.     

Suppression of human prostate cancer cell growth by forced expression of connexin genes

The cell-to-cell channels in gap junctions, formed of proteins called connexins (Cxs), provide a direct intercellular pathway for the passage of small signaling molecules (< or = 1 kD) between the cytoplasmic interiors of adjoining cells. It has been proposed that alteration in the expression and function of Cxs may be one of the genetic changes involved in the initiation of neoplasia. To elucidate the role of Cxs in the pathogenesis of human prostate cancer (PCA), the pattern of expression of Cx alpha 1 (Cx43) and Cx beta 1 (Cx32) was studied by immunocytochemical analysis in normal prostate and in prostate tumors of different histological grades. While normal prostate epithelial cells expressed only Cx beta 1, both Cx alpha 1 and Cx beta 1 were detected in PCA cells. The Cxs were localized at the cell-cell contact areas in normal prostate and well-differentiated prostate tumors; however, as prostate tumors progressed to more undifferentiated stages, the Cxs were localized in the cytoplasm, followed by an eventual loss in advanced stages. Thus, epithelial cells from prostate tumors showed subtle and gross alterations with regard to expression of Cx alpha 1 and Cx beta 1 and their assembly into gap junctions during the progression of PCA. Retroviral-mediated transfer of Cx alpha 1 and Cx beta 1 into a Cx-deficient human PCA cell line, LNCaP, inhibited growth, retarded tumorigenicity, and induced differentiation, and these effects were contingent upon the formation of gap junctions. In addition, the capacity to form gap junctions in most Cx-transduced LNCaP cells was lost upon serial passage. Taken together, these findings indicate that the control of proliferation and differentiation of epithelial cells in prostate tumors may depend on the appropriate assembly of Cx beta 1 and Cx alpha 1 into gap junctions and that the development of PCA may involve the positive selection of cells with an impaired ability to form gap junctions.     

Connexin and pannexin mediated cell-cell communication

In this review, we briefly summarize what is known about the properties of the three families of gap junction proteins, connexins, innexins and pannexins, emphasizing their importance as intercellular channels that provide ionic and metabolic coupling and as non-junctional channels that can function as a paracrine signaling pathway. We discuss that two distinct groups of proteins form gap junctions in deuterostomes (connexins) and protostomes (innexins), and that channels formed of the deuterostome homologues of innexins (pannexins) differ from connexin channels in terms of important structural features and activation properties. These differences indicate that the two families of gap junction proteins serve distinct, complementary functions in deuterostomes. In several tissues, including the CNS, both connexins and pannexins are involved in intercellular communication, but have different roles. Connexins mainly contribute by forming the intercellular gap junction channels, which provide for junctional coupling and define the communication compartments in the CNS. We also provide new data supporting the concept that pannexins form the non-junctional channels that play paracrine roles by releasing ATP and, thus, modulating the range of the intercellular Ca(2+)-wave transmission between astrocytes in culture.     

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

A new angle on blood–CNS interfaces: A role for connexins?

Neuronal signaling in the CNS depends on the microenvironment around synapses and axons. To prevent fluctuations in blood composition affecting the interstitial fluid and CSF, two barriers, the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), are interposed between the blood and the brain/CSF compartment. Brain capillary endothelial cells (ECs) constitute the BBB whereas choroid plexus epithelial (CPE) cells form the BCSFB. The anatomical basis of these barriers is located at the level of an intercellular junctional complex that impedes paracellular diffusion. Tight and adherens junctions are known as the principal constituents of this junctional complex. Transmembrane connexins (Cxs) are the prime building blocks of plasma membrane hemichannels that combine to form intercellular gap junctions (GJ). Although Cxs co-exist within the junctional complex, their influence on tight/adherens junctions and their role in barrier function of BBB ECs and CPE has been mostly ignored. Here, we review current knowledge on the role of Cxs in the BBB, BCSFB and other interfaces that subside within the CNS. We conclude that Cxs are a rather unexplored but promising target for influencing CNS barrier function.     

Identification of a potential regulator of the gap junction protein pannexin1

Recent studies have revealed a second class of gap-junction-forming proteins in vertebrates. These genes are termed pannexins, and it has been suggested that they perform similar functions as connexins. Pannexin1 is expressed in diverse tissues including the central nervous system and seems to form gap junction channels in the Xenopus oocyte expression system. Since protein interacting partners have frequently been described for connexins, the most prominent family of gap junction forming proteins, we thus started to search for candidate genes of pannexin interacting partners. Kvbeta3, a protein belonging to the family of regulatory beta-subunits of the voltage-dependent potassium channels, was identified as a binding partner of pannexin1 in an E. coli two-hybrid system. This result was verified by confocal laser scanning microscopy using double transfected Neuro2A cells. The colocalization of both proteins at the plasma membrane is suggestive of functional interaction.     

Cloning, embryonic expression, and functional characterization of two novel connexins from Xenopus laevis

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell-cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Passage of 17 kDa calmodulin through gap junctions of three vertebrate species

Gap junctions of some vertebrates are capable of passing the elongate molecule, calmodulin, with a molecular weight 8-17 times greater than the previously recognized size limits. Fluorescently labeled calmodulin (FCaM) (17.34 kDa) microinjected into oocytes of ovarian follicles from an amphibian, Xenopus laevis, and from two species of teleost fish, Danio rerio (Zebrafish) and Oryzias latipes (Medaka), is shown to transit their gap junctions and enter the surrounding epithelial cells. Passage of FCaM was terminated when follicles were first treated with 1 mM octanol, a molecule known to down-regulate gap junctions. There was no FCaM detected in the surrounding medium, nor did epithelial cells become fluorescent when follicles were incubated in medium containing dye. Calmodulin is well known to modulate many cytoplasmic reactions; thus, its passage through gap junctions opens possibilities of additional means by which cells may be supplied with this signaling molecule, and by which their supply may be regulated.     

Pannexin-1 as a potentiator of ligand-gated receptor signaling

Pannexins are a class of plasma membrane spanning proteins that presumably form a hexameric, non-selective ion channel. Although similar in secondary structure to the connexins, pannexins notably do not form endogenous gap junctions and act as bona fide ion channels. The pannexins have been primarily studied as ATP-release channels, but the overall diversity of their functions is still being elucidated. There is an intriguing theme with pannexins that has begun to develop. In this review we analyze several recent reports that converge on the idea that pannexin channels (namely Panx1) can potentiate ligand-gated receptor signaling. Although the literature remains sparse, this emerging concept appears consistent between both ionotropic and metabotropic receptors of several ligand families.     

Innexin and pannexin channels and their signaling

Innexins are bifunctional membrane proteins in invertebrates, forming gap junctions as well as non-junctional membrane channels (innexons). Their vertebrate analogues, the pannexins, have not only lost the ability to form gap junctions but are also prevented from it by glycosylation. Pannexins appear to form only non-junctional membrane channels (pannexons). The membrane channels formed by pannexins and innexins are similar in their biophysical and pharmacological properties. Innexons and pannexons are permeable to ATP, are present in glial cells, and are involved in activation of microglia by calcium waves in glia. Directional movement and accumulation of microglia following nerve injury, which has been studied in the leech which has unusually large glial cells, involves at least 3 signals: ATP is the “go” signal, NO is the “where” signal and arachidonic acid is a “stop” signal.     

Nature of plasmalemmal functional “hemichannels”

The molecular identity of the protein forming “hemichannels” at non-junctional membranes is disputed. The family of gap junction proteins, innexins, connexins, and pannexins share several common features, including permeability characteristics and sensitivity to blocking agents. Such overlap in properties renders the identification of which of these protein species actually establishes the non-junctional membrane conductance and permeability quite complicated, especially because in vertebrates pannexins and connexins have largely overlapping distributions in tissues. Recently, attempts to establish criteria to identify events that are “hemichannel” mediated and those to allow the distinction between connexin- from pannexin-mediated events have been proposed.Here, I present an update on that topic and discuss the most recent findings related to the nature of functional “hemichannels” focusing on connexin43 and pannexin1. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.