Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.     

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.     

Regulation of connexin‐ and pannexin‐based channels by post‐translational modifications

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi‐protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post‐translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N‐acetylation, S‐nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx‐channel activity and localisation.     

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H₂S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H₂S.     

Modulation of gap junction channels and hemichannels by growth factors

Gap junction hemichannels and cell-cell channels have roles in coordinating numerous cellular processes, due to their permeability to extra and intracellular signaling molecules. Another mechanism of cellular coordination is provided by a vast array of growth factors that interact with relatively selective cell membrane receptors. These receptors can affect cellular transduction pathways, including alteration of intracellular concentration of free Ca(2+) and free radicals and activation of protein kinases or phosphatases. Connexin and pannexin based channels constitute recently described targets of growth factor signal transduction pathways, but little is known regarding the effects of growth factor signaling on pannexin based channels. The effects of growth factors on these two channel types seem to depend on the cell type, cell stage and connexin and pannexin isoform expressed. The functional state of hemichannels and gap junction channels are affected in opposite directions by FGF-1 via protein kinase-dependent mechanisms. These changes are largely explained by channels insertion in or withdrawal from the cell membrane, but changes in open probability might also occur due to changes in phosphorylation and redox state of channel subunits. The functional consequence of variation in cell-cell communication via these membrane channels is implicated in disease as well as normal cellular responses.     

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca2+ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.     

Connexin- and Pannexin-Based Channels in Normal Skeletal Muscles and Their Possible Role in Muscle Atrophy

Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca²⁺ concentration and release of diverse metabolites (e.g., NAD⁺ and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.     

Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇ receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇ expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.     

Hemichannels: permeants and their effect on development, physiology and death

Hemichannels, which are one half of the gap junction channels, have independent physiological roles. Although hemichannels consisting of connexins are more widely documented, hemichannels of pannexins, proteins homologous to invertebrate gap junction proteins also have been studied. There are at least 21 different connexin and three pannexin isotypes. This variety in isotypes results in tissue-specific hemichannels, which have been implicated in varied events ranging from development, cell survival, to cell death. Hemichannel function varies with its spatio-temporal opening, thus demanding a refined degree of regulation. This review discusses the activity of hemichannels and the molecules released in different physiological states and their impact on tissue functioning.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Connexin and pannexin hemichannels of neurons and astrocytes

Hemichannels are large pore ion channels that in the traditional view are formed when half a gap connexin junction opens to the extracellular space. It is now evident that other ion channel families, including the newly discovered pannexin family can form channels with all the nascent properties of hemichannels. This suggests that hemichannels should now be defined to include members of non-connexin families. Several connexin, and two pannexins are expressed in neurons and astrocytes where they may function in release of ATP and glutamate. Additionally, pannexin-1 appears to play a role in neuronal death. Hemichannels form a novel and unique class of ion channels that likely have diverse physiological and pathophysiological roles in the nervous system.     

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Calcium and connexin-based intercellular communication, a deadly catch?

Ca²⁺ is known as a universal messenger mediating a wide variety of cellular processes, including cell death. In fact, this ion has been proposed as the ‘cell death master’, not only at the intracellular but also at the intercellular level. The most direct form of intercellular spread of cell death is mediated by gap junction channels. These channels have been shown to propagate cell death as well as cell survival signals between the cytoplasm of neighbouring cells, reflecting the dual role of Ca²⁺ signals, i.e. cell death versus survival. Its precursor, the unopposed hemichannel (half of a gap junction channel), has recently joined in as a toxic pore connecting the intracellular with the extracellular environment and allowing the passage of a range of substances. The biochemical nature of the so-called intercellular cell death molecule, transferred through gap junctions or released/taken up via hemichannels, remains elusive but several studies pinpoint Ca²⁺ itself or its messenger inositol trisphosphate as the responsible masters in crime. Although direct evidence is still lacking, indirect data including Ca²⁺ involvement in intercellular communication and cell death, and effects of intercellular communication on intracellular Ca²⁺ homeostasis, support this hypothesis. In addition, hemichannels and their molecular building blocks, connexin or pannexin proteins, may exert their effects on Ca²⁺-dependent cell death at the intracellular level, independently from their channel functions. This review provides a cutting edge overview of the current knowledge and underscores the intimate connection between intercellular communication, Ca²⁺ signalling and cell death.     

Connexin and pannexin hemichannels of neurons and astrocytes

Hemichannels are large pore ion channels that in the traditional view are formed when half a gap connexin junction opens to the extracellular space. It is now evident that other ion channel families, including the newly discovered pannexin family can form channels with all the nascent properties of hemichannels. This suggests that hemichannels should now be defined to include members of non-connexin families. Several connexin, and two pannexins are expressed in neurons and astrocytes where they may function in release of ATP and glutamate. Additionally, pannexin-1 appears to play a role in neuronal death. Hemichannels form a novel and unique class of ion channels that likely have diverse physiological and pathophysiological roles in the nervous system.     

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.     

Connexins: substrates and regulators of autophagy

Connexins mediate intercellular communication by assembling into hexameric channel complexes that act as hemichannels and gap junction channels. Most connexins are characterized by a very rapid turn-over in a variety of cell systems. The regulation of connexin turn-over by phosphorylation and ubiquitination events has been well documented. Moreover, different pathways have been implicated in connexin degradation, including proteasomal and lysosomal-based pathways. Only recently, autophagy emerged as an important connexin-degradation pathway for different connexin isoforms. As such, conditions well known to induce autophagy have an immediate impact on the connexin-expression levels. This is not only limited to experimental conditions but also several pathophysiological conditions associated with autophagy (dys)function affect connexin levels and their presence at the cell surface as gap junctions. Finally, connexins are not only substrates of autophagy but also emerge as regulators of the autophagy process. In particular, several connexin isoforms appear to recruit pre-autophagosomal autophagy-related proteins, including Atg16 and PI3K-complex components, to the plasma membrane, thereby limiting their availability and capacity for regulating autophagy.

     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

Hunting for connexin hemichannels

Connexin hemichannels (connexons) are building blocks of gap junctions but also function as free unapposed channels, which has become an active field of research. Defining functions of hemichannels and their involvement in any biological event requires ruling out possible participation of other channels that share biophysical and regulatory properties, for example pannexins, CALHM1 and P2X receptors. The lack of specific inhibitors for these channels has become an obstacle in elucidating the role of connexin hemichannels. Several experimental approaches are now available to identify hemichannels at the cell surface and to characterize their electrophysiological, permeability and regulatory properties. The use of connexin knockout/knockdown, and the development of peptides that target intracellular connexin domains and specific antibodies directed to extracellular domains have helped to dissect the role of hemichannels in endogenously expressing systems. Moreover, studies of connexin mutants in exogenous expression systems have provided convincing evidence on hemichannels in the pathogenesis of several human genetic diseases. We here present a brief overview of connexin hemichannels as functional channels and itemize a list of aspects to consider when concluding on their involvement.     

Structure,Regulation and Function of Gap Junctions in Liver

Abstract

Gap junctions are a specialized group of cell-to-cell junctions that mediate direct intercellular communication between cells. They arise from the interaction of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. In liver, gap junctions are predominantly found in hepatocytes and play critical roles in virtually all phases of the hepatic life cycle, including cell growth, differentiation, liver-specific functionality and cell death. Liver gap junctions are directed through a broad variety of mechanisms ranging from epigenetic control of connexin expression to post-translational regulation of gap junction activity. This paper reviews established and novel aspects regarding the architecture, control and functional relevance of liver gap junctions.     

Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions

Gap junctions are intercellular channels that connect the cytoplasms of adjacent cells. For gap junctions to properly control organ formation and electrical synchronization in the heart and the brain, connexin-based hemichannels must be correctly targeted to cell-cell borders. While it is generally accepted that gap junctions form via lateral diffusion of hemichannels following microtubule-mediated delivery to the plasma membrane, we provide evidence for direct targeting of hemichannels to cell-cell junctions through a pathway that is dependent on microtubules; through the adherens-junction proteins N-cadherin and beta-catenin; through the microtubule plus-end-tracking protein (+TIP) EB1; and through its interacting protein p150(Glued). Based on live cell microscopy that includes fluorescence recovery after photobleaching (FRAP), total internal reflection fluorescence (TIRF), deconvolution, and siRNA knockdown, we propose that preferential tethering of microtubule plus ends at the adherens junction promotes delivery of connexin hemichannels directly to the cell-cell border. These findings support an unanticipated mechanism for protein delivery to points of cell-cell contact.