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.

Decreased connexin 40 expression of the sinoatrial node mediates ischemic stroke-induced arrhythmia in mice.

BACKGROUND: Arrhythmia is the most common cardiac complication after ischemic stroke. Connexin 40 is the staple component of gap junctions, which influences the propagation of cardiac electrical signals in the sinoatrial node. However, the role of connexin 40 in post-stroke arrhythmia remains unclear. METHODS: In this study, a permanent middle cerebral artery occlusion model was used to simulate the occurrence of an ischemic stroke. Subsequently, an electrocardiogram was utilized to record and assess variations in electrocardiogram measures. In addition, optical tissue clearing and whole-mount immunofluorescence staining were used to confirm the anatomical localization of the sinoatrial node, and the sinoatrial node tissue was collected for RNA sequencing to screen for potential pathological mechanisms. Lastly, the rAAV9-Gja5 virus was injected with ultrasound guidance into the heart to increase Cx40 expression in the sinoatrial node. RESULTS: We demonstrated that the mice suffering from a permanent middle cerebral artery occlusion displayed significant arrhythmia, including atrial fibrillation, premature ventricular contractions, atrioventricular block, and abnormal electrocardiogram parameters. Of note, we observed a decrease in connexin 40 expression within the sinoatrial node after the ischemic stroke via RNA sequencing and western blot. Furthermore, rAAV9-Gja5 treatment ameliorated the occurrence of arrhythmia following stroke. CONCLUSIONS: In conclusion, decreased connexin 40 expression in the sinoatrial node contributed to the ischemic stroke-induced cardiac arrhythmia. Therefore, enhancing connexin 40 expression holds promise as a potential therapeutic approach for ischemic stroke-induced arrhythmia.

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.

Pannexin 1 dysregulation in Duchenne muscular dystrophy and its exacerbation of dystrophic features in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. METHODS: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1-/-/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. RESULTS: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1-/-/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. CONCLUSIONS: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.

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.

Measurement of Ca2+ Uptake Through Connexin Hemichannels.

Increasing evidence points to deregulated flux of ionized calcium (Ca2+) mediated by hyperactive mutant connexin (Cx) hemichannels (HCs) as a common gain-of-function etiopathogenetic mechanism for several diseases, ranging from skin disorders to nervous system defects. Furthermore, the opening of nonmutated Cx HCs is associated with an impressive list of widespread diseases including, but not limited to, ischemia/stroke, Alzheimer's disease, and epilepsy. HC inhibitors are attracting a growing attention due to their therapeutic potential for numerous pathologies. This chapter describes a quantitative method to measure Ca2+ uptake though HCs expressed in cultured cells. The assay we developed can be used to probe HC activity as wells as to test HC inhibitors. Furthermore, with minor changes it can be easily adapted to high-throughput high-content platforms and/or primary cells and microtissues.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase.

The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.

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.

Connexin 43 Prevents Radiation-Induced Intestinal Damage via the Ca2+-Dependent PI3K/Akt Signaling Pathway.

Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.

Simulation of gap junction formation reveals critical role of Cys disulfide redox state in connexin hemichannel docking.

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