Pannexins in the musculoskeletal system: new targets for development and disease progression.

During cell differentiation, growth, and development, cells can respond to extracellular stimuli through communication channels. Pannexin (Panx) family and connexin (Cx) family are two important types of channel-forming proteins. Panx family contains three members (Panx1-3) and is expressed widely in bone, cartilage and muscle. Although there is no sequence homology between Panx family and Cx family, they exhibit similar configurations and functions. Similar to Cxs, the key roles of Panxs in the maintenance of physiological functions of the musculoskeletal system and disease progression were gradually revealed later. Here, we seek to elucidate the structure of Panxs and their roles in regulating processes such as osteogenesis, chondrogenesis, and muscle growth. We also focus on the comparison between Cx and Panx. As a new key target, Panxs expression imbalance and dysfunction in muscle and the therapeutic potentials of Panxs in joint diseases are also discussed.

A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

The Role of the Gap Junction Protein Connexin in Adrenal Gland Tumorigenesis.

Gap junctions (GJs) are important in the regulation of cell growth, morphology, differentiation and migration. However, recently, more attention has been paid to their role in the pathogenesis of different diseases as well as tumorigenesis, invasion and metastases. The expression pattern and possible role of connexins (Cxs), as major GJ proteins, under both physiological and pathological conditions in the adrenal gland, were evaluated in this review. The databases Web of Science, PubMed and Scopus were searched. Studies were evaluated if they provided data regarding the connexin expression pattern in the adrenal gland, despite current knowledge of this topic not being widely investigated. Connexin expression in the adrenal gland differs according to different parts of the gland and depends on ACTH release. Cx43 is the most studied connexin expressed in the adrenal gland cortex. In addition, Cx26, Cx32 and Cx50 were also investigated in the human adrenal gland. Cx50 as the most widespread connexin, along with Cx26, Cx29, Cx32, Cx36 and Cx43, has been expressed in the adrenal medulla with distinct cellular distribution. Considerable effort has recently been directed toward connexins as therapeutically targeted molecules. At present, there exist several viable strategies in the development of potential connexin-based therapeutics. The differential and hormone-dependent distribution of gap junctions within adrenal glands, the relatively large gap junction within this gland and the increase in the gap junction size and number following hormonal treatment would indicate that gap junctions play a pivotal role in cell functioning in the adrenal gland.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

BACKGROUND: Fibrosis results from excessive scar formation after tissue injury. Injured cells release alarmins such as interleukin 1 (IL-1) α and ß as primary mediators initiating tissue repair. However, how alarmins from different cell types differentially regulate fibrosis remains to be explored. METHODS: Here, we used tissue specific knockout strategy to illustrate a unique contribution of endothelial cell-derived IL-1α to lung and liver fibrosis. The two fibrotic animal model triggered by bleomycin and CCl4 were used to study the effects of endothelial paracrine/angiocrine IL-1α in fibrotic progression. Human umbilical vein endothelial cells (HUVEC) were performed to explore the production of angiocrine IL-1α at both transcriptional and post-transcriptional levels in vitro. RESULTS: We found that endothelial paracrine/angiocrine IL-1α primarily promotes lung and liver fibrosis during the early phase of organ repair. By contrast, myeloid cell-specific ablation of IL-1α in mice resulted in little influence on fibrosis, suggesting the specific pro-fibrotic role of IL-1α from endothelial cell but not macrophage. In vitro study revealed a coordinated regulation of IL-1α production in human primary endothelial cells at both transcriptional and post-transcriptional levels. Specifically, the transcription of IL-1α is regulated by RIPK1, and after caspase-8 (CASP8) cleaves the precursor form of IL-1α, its secretion is triggered by ion channel Pannexin 1 upon CASP8 cleavage. CONCLUSIONS: Endothelial cell-produced IL-1α plays a unique role in promoting organ fibrosis. Furthermore, the release of this angiocrine alarmin relies on a unique molecular mechanism involving RIPK1, CASP8, and ion channel Pannexin 1.

Ibrutinib Contributes to Atrial Arrhythmia through the Autophagic Degradation of Connexins by Inhibiting the PI3K-AKT-mTOR Signaling Pathway.

BACKGROUND: Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated. METHODS: We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation. RESULTS: About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40. CONCLUSIONS: The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy. CLINICAL TRIAL REGISTRATION: ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.

Astroglial gap junctions strengthen hippocampal network activity by sustaining afterhyperpolarization via KCNQ channels.

Throughout the brain, astrocytes form networks mediated by gap junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecular gap junction channels shape neuronal network patterns remains unclear. Here, using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we show that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a facilitation-depression model, we identify neuronal afterhyperpolarization as the key parameter underlying bursting pattern regulation by extracellular potassium in mice with disconnected astrocytes. We confirm this prediction experimentally and reveal that astroglial network control of extracellular potassium sustains neuronal afterhyperpolarization via KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap junctions mechanistically strengthen neuronal population bursts and point to approaches for controlling aberrant activity in neurological diseases.

GJB3: a comprehensive biomarker in pan-cancer prognosis and immunotherapy prediction.

BACKGROUND: A wide range of connexins are situated between normal-normal cells, cancer-cancer cells, and cancer-normal cells. Abnormalities in connexin expression are typically accompanied by cancer development; however, no systematic studies have examined the role of Gap Junction Protein Beta 3 (GJB3) in the context of tumor progression and immunity, especially when considering a broad range of cancer types. METHODS: In this study, data on GJB3 expression were gathered from Genotype-Tissue Expression, Cancer Cell Line Encyclopedia, and The Cancer Genome Atlas databases. Then, we analyzed the relationship between GJB3 expression and tumor characteristics. In vitro experiments using colony formation, EdU, CCK8, transwell migration assays, immunohistochemistry and western blot were performed to investigate the function of GJB3 in tumor progression of various cell lines. A drug sensitivity analysis of GJB3 was performed using the Genomics of Drug Sensitivity in Cancer database. RESULT: Our findings demonstrate that GJB3 is widely expressed in various cancers and correlates significantly with disease stages, patient survival, immunotherapy response, and pharmaceutical guidance. Additionally, GJB3 plays a role in different cancer pathways, as well as in different immune and molecular subtypes of cancer. Co-expression of GJB3 with immune checkpoint genes was observed. Further experiments showed that knockdown of GJB3 inhibited the PI3K/AKT pathway and resulted in reduced proliferation, migration, and viability of different cancer cells. CONCLUSION: Overall, GJB3 shows potential as a molecular biomarker and therapeutic target for various cancers, particularly lung adenocarcinomas, mesothelioma, pancreatic adenocarcinoma. Thus, GJB3 may represent a new therapeutic target for a wide range of cancers.

Human pannexin 1 channel is not phosphorylated by Src tyrosine kinase at Tyr199 and Tyr309.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Molecular Dynamics Simulation of Permeation Through Connexin Channels.

Molecular dynamics (MD) simulations are a collection of computational tools that can be used to trace intermolecular interactions at the sub-nanometer level. They offer possibilities that are often unavailable to experimental methods, making MD an ideal complementary technique for the understanding a plethora of biological processes. Thanks to significant efforts by many groups of developers around the world, setting up and running MD simulations has become progressively simpler. However, simulating ionic permeation through membrane channels still presents significant caveats.MD simulations of connexin (Cx) hemichannels (HCs) are particularly problematic because HCs create wide pores in the plasma membrane, and the lateral sizes of the extracellular and intracellular regions are quite different. In this chapter, we provide a detailed instruction to perform MD simulations aimed at computationally modeling the permeation of inorganic ions and larger molecules through Cx HCs.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

A Dye Uptake Assay to Measure Large-Pore Channel Activity in Trypanosoma cruzi.

Large-pore channels allow the exchange of ions and molecules between the intra- and extracellular compartments. These channels are structures formed by several protein families with little or no evolutionary linkages that include connexins (Cxs), pannexins (Panxs), innexins (Inxs), CALHM1, and LRRC8 proteins. Recently, we have described the unnexins (Unxs) proteins expressed in Trypanosoma cruzi (T. cruzi) that also is like to form large-pore channels at the plasma membrane. In this chapter, we describe a dye uptake method for evaluating the unnexin-formed channel function in T. cruzi, as well as the methods for evaluating their participation in the transformation of trypomastigotes into amastigotes. These methods can facilitate understanding the role of large-pore channels in the parasite's biology.

Protocol for the Study of Connexin and DNA Interactions.

Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, "noncanonical" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.