Role of connexins and pannexins during ontogeny,regeneration, and pathologies of bone

Electron micrographs revealed the presence of gap junctions in osteoblastic cells over 40 years ago. These intercellular channels formed from connexins are present in bone forming osteoblasts, bone resorbing osteoclasts, and osteocytes (mature osteoblasts embedded in the mineralized bone matrix). More recently, genetic and pharmacologic studies revealed the role of connexins, and in particular Cx43, in the differentiation and function of all bone types. Furthermore, mutations in the gene encoding Cx43 were found to be causally linked to oculodentodigital dysplasia, a condition that results in an abnormal skeleton. Pannexins, molecules with similar structure and single-membrane channel forming potential as connexins when organized as hemichannels, are also expressed in osteoblastic cells. The function of pannexins in bone and cartilage is beginning to be uncovered, but more research is needed to determine the role of pannexins in bone development, adult bone mass and skeletal homeostasis. We describe here the current knowledge on the role of connexins and pannexins on skeletal health and disease.

     

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.     

Role of connexins and pannexins during ontogeny,regeneration, and pathologies of bone

Electron micrographs revealed the presence of gap junctions in osteoblastic cells over 40 years ago. These intercellular channels formed from connexins are present in bone forming osteoblasts, bone resorbing osteoclasts, and osteocytes (mature osteoblasts embedded in the mineralized bone matrix). More recently, genetic and pharmacologic studies revealed the role of connexins, and in particular Cx43, in the differentiation and function of all bone types. Furthermore, mutations in the gene encoding Cx43 were found to be causally linked to oculodentodigital dysplasia, a condition that results in an abnormal skeleton. Pannexins, molecules with similar structure and single-membrane channel forming potential as connexins when organized as hemichannels, are also expressed in osteoblastic cells. The function of pannexins in bone and cartilage is beginning to be uncovered, but more research is needed to determine the role of pannexins in bone development, adult bone mass and skeletal homeostasis. We describe here the current knowledge on the role of connexins and pannexins on skeletal health and disease.     

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.     

溶酶体跨膜蛋白175钾通道蛋白质与帕金森病

帕金森病(Parkinson’s disease,PD)是一种复杂的神经退行性疾病,以运动障碍和非运动症状为特征。虽然以多巴胺为基础的疗法在疾病的早期阶段能有效地对抗症状,但缺乏神经保护药物意味着疾病仍在继续发展。PD病人迫切需要新的疾病修饰疗法和新的治疗策略。遗传学研究表明,罕见和常见的基因变异都会导致PD的发生和发展。跨膜蛋白175(transmembrane protein 175,TMEM175)是来自溶酶体的PD风险基因之一,编码一种具有新型结构的溶酶体钾通道蛋白质,参与维持溶酶体膜电位和pH的稳定性。随着对TMEM175的了解,多项研究证实TMEM175的缺陷能够导致溶酶体功能障碍,诱导神经元α-突触核蛋白(α-synuclein)的病理性聚集,促进帕金森病发生。鉴于溶酶体TMEM175通道蛋白的重要功能,TMEM175基因可能是帕金森病及其他神经退行性疾病治疗的潜在靶点。本文综述了溶酶体TMEM175基因的结构及功能,重点阐述了其作为溶酶体内稳态调节因子通过影响溶酶体的功能参与PD发生发展的过程,并对其研究进行了展望。以TMEM175为靶标,筛选具有保持TMEM175的活性状态或增强其表达的药物可能具有改善PD病人病情的效果,但TMEM175如何通过保持其通道的开放和闭合状态之间的平衡来调节溶酶体的离子稳态,具体机制尚需要进一步研究探讨。未来对该离子通道蛋白质的进一步研究将为 靶向PD治疗带来新的策略和思路,为在PD的诊断与治疗中奠定TMEM175的靶标分子地位提供支持。     

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.     

Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes

Several new findings have emphasized the role of neuron-specific gap junction proteins (connexins) and electrical synapses in processing sensory information and in synchronizing the activity of neuronal networks. We have recently shown that pannexins constitute an additional family of proteins that can form gap junction channels in a heterologous expression system and are also widely expressed in distinct neuronal populations in the brain, where they may represent a novel class of electrical synapses. In this study, we have exploited the hemichannel-forming properties of pannexins to investigate their sensitivity to well-known connexin blockers. By combining biochemical and electrophysiological approaches, we report here further evidence for the interaction of pannexin1 (Px1) with Px2 and demonstrate that the pharmacological sensitivity of heteromeric Px1/Px2 is similar to that of homomeric Px1 channels. In contrast to most connexins, both Px1 and Px1/Px2 hemichannels were not gated by external Ca2+. In addition, they exhibited a remarkable sensitivity to blockade by carbenoxolone (with an IC50 of approximately 5 microm), whereas flufenamic acid exerted only a modest inhibitory effect. The opposite was true in the case of connexin46 (Cx46), thus indicating that gap junction blockers are able to selectively modulate pannexin and connexin channels.     

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.     

SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.     

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.     

ATP transporters in the joints

Extracellular adenosine triphosphate (ATP) plays a central role in a wide variety of joint diseases. ATP is generated intracellularly, and the concentration of the extracellular ATP pool is determined by the regulation of its transport out of the cell. A variety of ATP transporters have been described, with connexins and pannexins the most commonly cited. Both form intercellular channels, known as gap junctions, that facilitate the transport of various small molecules between cells and mediate cell–cell communication. Connexins and pannexins also form pores, or hemichannels, that are permeable to certain molecules, including ATP. All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathological conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA), indicating that they may be involved in the onset and progression of these pathologies. The aging of the global population, along with increases in the prevalence of obesity and metabolic dysfunction, is associated with a rising frequency of joint diseases along with the increased costs and burden of related illness. The modulation of connexins and pannexins represents an attractive therapeutic target in joint disease, but their complex regulation, their combination of gap-junction-dependent and -independent functions, and their interplay between gap junction and hemichannel formation are not yet fully elucidated. In this review, we try to shed light on the regulation of these proteins and their roles in ATP transport to the extracellular space in the context of joint disease, and specifically OA and RA.     

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.     

Multiple and complex influences of connexins and pannexins on cell death

Cell death is a fundamental process for organogenesis, immunity and cell renewal. During the last decades a broad range of molecular tools were identified as important players for several different cell death pathways (apoptosis, pyroptosis, necrosis, autosis…). Aside from these direct regulators of cell death programs, several lines of evidence proposed connexins and pannexins as potent effectors of cell death. In the present review we discussed the potential roles played by connexins, pannexins and innexins in the different cell death programs at different scales from gap junction intercellular communication to protein–protein interactions. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Mechanisms of ATP release in pain: role of pannexin and connexin channels

Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.     

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.     

Pannexin channel and connexin hemichannel expression in vascular function and inflammation

Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.     

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.     

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.     

Biological role of connexin intercellular channels and hemichannels

Gap junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.     

Probenecid, a gout remedy, inhibits pannexin 1 channels

Probenecid is a well-established drug for the treatment of gout and is thought to act on an organic anion transporter, thereby affecting uric acid excretion in the kidney by blocking urate reuptake. Probenecid also has been shown to affect ATP release, leading to the suggestion that ATP release involves an organic anion transporter. Other pharmacological evidence and the observation of dye uptake, however, suggest that the nonvesicular release of ATP is mediated by large membrane channels, with pannexin 1 being a prominent candidate. In the present study we show that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus probenecid allows for discrimination between channels formed by connexins and pannexins.