The life cycle of a connexin: Gap junction formation,removal, and degradation

Gap junction proteins, connexins, possess many properties that are atypical of other well-characterized integral membrane proteins. Oligomerization of connexins into hemichannels (connexons) has been shown to occur after the protein exits the endoplasmic reticulum. Once delivered to the cell surface, connexons from one cell pair with connexons from a neighboring cell, a process that is facilitated by calcium-dependent cell adhesion molecules. Channels cluster into defined plasma membrane domains to form plaques. Unexpectedly, gap junctions are not stable (half-life <5 h) and are thought to be retrieved back into the cell in the form of double membrane structures when one cell internalizes the entire gap junction through endocytosis. Evidence exists for both proteasomal and lysosomal degradation of gap junctions, and it remains possible that both mechanisms are involved in connexin degradation. In addition to opening and closing of gap junction channels (gating), the formation and removal of gap junctions play an essential role in regulating the level of intercellular communication.     

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.     

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.     

Endocytosis and post-endocytic sorting of connexins

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells in solid tissues to directly exchange ions and small molecules. These channels assemble into distinct plasma membrane domains known as gap junctions. Gap junction intercellular communication plays critical roles in numerous cellular processes, including control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are dynamic plasma membrane domains, and there is increasing evidence that modulation of endocytosis and post-endocytic trafficking of connexins are important mechanisms for regulating the level of functional gap junctions at the plasma membrane. The emerging picture is that multiple pathways exist for endocytosis and sorting of connexins to lysosomes, and that these pathways are differentially regulated in response to physiological and pathophysiological stimuli. Recent studies suggest that endocytosis and lysosomal degradation of connexins is controlled by a complex interplay between phosphorylation and ubiquitination. This review summarizes recent progress in understanding the molecular mechanisms involved in endocytosis and post-endocytic sorting of connexins, and the relevance of these processes to the regulation of gap junction intercellular communication under normal and pathophysiological conditions. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

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.     

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.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Enhanced neurite outgrowth in PC12 cells mediated by connexin hemichannels and ATP

Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.     

Connexins and their channels in cell growth and cell death

Direct communication between cells, mediated by gap junctions, is nowadays considered as an indispensable mechanism in the maintenance of cellular homeostasis. In fact, gap junctional intercellular communication is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death. For a long time, it was believed that this was merely a result of the capacity of gap junctions to control the direct intercellular exchange of essential cellular messengers. However, recent data show that the picture is more complicated than initially thought, as structural precursors of gap junctions, connexins and gap junction hemichannels, can affect the cellular homeostatic balance independently of gap junctional intercellular communication. In this paper, we summarize the current knowledge concerning the roles of connexins and their channels in the control of cellular homeostasis, with the emphasis on cell growth and cell death. We also briefly discuss the role of gap junctional intercellular communication in carcinogenesis and the potential use of connexins as tools for cancer therapy.     

Connexins,gap junctions and tissue invasion

Formation of metastases negatively impacts the survival prognosis of cancer patients. Globally, if the various steps involved in their formation are relatively well identified, the molecular mechanisms responsible for the emergence of invasive cancer cells are still incompletely resolved. Elucidating what are the mechanisms that allow cancer cells to evade from the tumor is a crucial point since it is the first step of the metastatic potential of a solid tumor. In order to be invasive, cancer cells have to undergo transformations such as down-regulation of cell-cell adhesions, modification of cell-matrix adhesions and acquisition of proteolytic properties. These transformations are accompanied by the capacity to “activate” stromal cells, which may favor the motility of the invasive cells through the extracellular matrix. Since modulation of gap junctional intercellular communication is known to be involved in cancer, we were interested to consider whether these different transformations necessary for the acquisition of invasive phenotype are related with gap junctions and their structural proteins, the connexins. In this review, emerging roles of connexins and gap junctions in the process of tissue invasion are proposed.     

Gap junction- and hemichannel-independent actions of connexins

Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.     

Gap junctions and neurological disorders of the central nervous system

Gap junctions are intercellular channels which directly connect the cytoplasm between neighboring cells. In the central nervous system (CNS) various kinds of cells are coupled by gap junctions, which play an important role in maintaining normal function. Neuronal gap junctions are involved in electrical coupling and may also contribute to the recovery of function after cell injury. Astrocytes are involved in the pathology of most neuronal disorders, including brain ischemia, Alzheimer's disease and epilepsy. In the pathology of brain tumors, gap junctions may be related to the degree of malignancy and metastasis. However, the role of connexins, gap junctions and hemichannels in the pathology of the diseases in the CNS is still ambiguous. Of increasing importance is the unraveling of the function of gap junctions in the neural cell network, involving neurons, astrocytes, microglia and oligodendrocytes. A better understanding of the role of gap junctions may contribute to the development of new therapeutic approaches to treating diseases of the CNS.     

Gap junction- and hemichannel-independent actions of connexins

Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.     

Connexins: a myriad of functions extending beyond assembly of gap junction channels

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.     

Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

This review is based in part on a roundtable discussion session: “Physiological roles for heterotypic/heteromeric channels” at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.     

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.     

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.     

Gap junctions and hemichannels: communicating cell death in neurodevelopment and disease

Gap junctions are unique membrane channels that play a significant role in intercellular communication in the developing and mature central nervous system (CNS). These channels are composed of connexin proteins that oligomerize into hexamers to form connexons or hemichannels. Many different connexins are expressed in the CNS, with some specificity with regard to the cell types in which distinct connexins are found, as well as the timepoints when they are expressed in the developing and mature CNS. Both the main neuronal Cx36 and glial Cx43 play critical roles in neurodevelopment. These connexins also mediate distinct aspects of the CNS response to pathological conditions. An imbalance in the expression, translation, trafficking and turnover of connexins, as well as mutations of connexins, can impact their function in the context of cell death in neurodevelopment and disease. With the ever-increasing understanding of connexins in the brain, therapeutic strategies could be developed to target these membrane channels in various neurological disorders.     

Regulation of gap junction intercellular communication by the ubiquitin system

Intercellular communication via gap junctions plays a critical role in numerous cellular processes, including the control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are aggregates of intercellular channels that enable adjacent cells in solid tissues to directly exchange ions and small molecules. These channels are formed by a family of integral membrane proteins called connexins, of which the best studied is connexin43. Connexins have a high turnover rate in most tissue types, and degradation of connexins is considered to be a tightly regulated process. Post-translational modification of connexins by ubiquitin is emerging as an important event in the regulation of connexin degradation. Ubiquitination is involved in endoplasmic reticulum-associated degradation of connexins as well as in trafficking of connexins to lysosomes. At both the endoplasmic reticulum and the plasma membrane, ubiquitination of connexins is strongly affected by changes in the extracellular environment. There is increasing evidence that the regulation of connexin ubiquitination might be an important mechanism for rapidly modifying the level of functional gap junctions at the plasma membrane, under both normal and pathological conditions. This review discusses the current knowledge about the regulation of intercellular communication via gap junctions by ubiquitination of connexins.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.