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.

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.

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.

Identification of novel and known genetic variants associated with hereditary hearing loss in iranian families using whole exome sequencing.

BACKGROUND: Hearing loss (HL) is a common sensory impairment worldwide, with genetic and environmental factors contributing to its occurrence. Next Generation Sequencing (NGS) plays a crucial role in identifying the genetic factors involved in this heterogeneous disorder. METHODS AND RESULTS: In this study, a total of 9 unrelated Iranian families, each having at least one affected individual who tested negative for mutations in GJB2, underwent screening using whole exome sequencing (WES). The pathogenicity and novelty of the identified variant was checked using various databases. Co-segregation study was also performed to confirm the presence of the candidate variants in parents. Plus, The pathogenicity of the detected variant was assessed through in silico analysis using a number of mutation prediction software tools. Among the 9 investigated families, hearing loss-causing genes were identified in 6 families. the mutations were observed in USH2A, CLRN1, BSND, SLC26A4, and MITF, with two of the identified mutations being novel. CONCLUSION: Discovering additional variants and broadening the range of mutations associated with hearing impairment has the potential to enhance the diagnostic effectiveness of molecular testing in patient screening, and can also lead to improved counseling aimed at reducing the risk of affected offspring for high-risk couples.

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.

Unusual phenotype in 35delG mutation: a case report.

BACKGROUND: Mutations in the GJB2 gene, which encodes the protein connexin 26 and is involved in inner ear homeostasis, are identified in approximately 50% of patients with autosomal recessive nonsyndromic hearing loss, making it one of the primary causes of prelingual nonsyndromic hearing loss in various populations. The 35delG mutation, one of the most common mutations of the GJB2 gene, usually causes prelingual, bilateral mild to profound, nonprogressive sensorineural hearing loss. CASE PRESENTATION: We present an unusual case of an 18-year-old Turkish female with heterozygous 35delG mutation and postlingual, profound-sloping, progressive and fluctuating unilateral sensorineural hearing loss. The phenotype is different from the usual findings. CONCLUSIONS: The 35delG mutation causing hearing loss may not always be reflected in the phenotype as expected and therefore may have different audiologic manifestations.

Comparison of vestibular function in hereditary hearing loss patients with GJB2, CDH23, and SLC26A4 variants.

To investigate the association between hereditary hearing loss and vestibular function, we compared vestibular function and symptoms among patients with GJB2, SLC26A4, and CDH23 variants. Thirty-nine patients with sensory neural hearing loss (11 males and 28 females) with biallelic pathogenic variants in either GJB2, SLC26A4, or CDH23 were included in this study (13 GJB2, 15 SLC26A4, and 11 CDH23). The patients were examined using caloric testing and cervical and ocular vestibular-evoked myogenic potentials (cVEMP and oVEMP). We also compared vestibular function and symptoms between patients with these gene variants and 78 normal-hearing ears without vestibular symptoms as controls. The frequency of semicircular canal hypofunction in caloric testing was higher in patients with SLC26A4 variants (47%) than in those with GJB2 (0%) and CDH23 variants (27%). According to the cVEMP results, 69% of patients with GJB2 variants had saccular hypofunction, a significantly higher proportion than in those carrying other variants (SLC26A4, 20%; CDH23, 18%). In oVEMP, which reflects utricular function, no difference was observed in the frequency of hypofunction among the three genes (GJB2, 15%; SLC26A4, 40%; and CDH23, 36%). Hence, discernable trends indicate vestibular dysfunction associated with each gene.

Structural basis for pathogenic variants of GJB2 and hearing levels of patients with hearing loss.

OBJECTIVES: The crystal structure of the six protomers of gap junction protein beta 2 (GJB2) enables prediction of the effect(s) of an amino acid substitution, thereby facilitating investigation of molecular pathogenesis of missense variants of GJB2. This study mainly focused on R143W variant that causes hearing loss, and investigated the relationship between amino acid substitution and 3-D structural changes in GJB2. METHODS: Patients with nonsyndromic hearing loss who appeared to have two GJB2 pathogenic variants, including the R143W variant, were investigated. Because the X-ray crystal structure of the six protomers of the GJB2 protein is known, R143W and structurally related variants of GJB2 were modeled using this crystal structure as a template. The wild-type crystal structure and the variant computer-aided model were observed and the differences in molecular interactions within the two were analyzed. RESULTS: The predicted structure demonstrated that the hydrogen bond between R143 and N206 was important for the stability of the protomer structure. From this prediction, R143W related N206S and N206T variants showed loss of the hydrogen bond. CONCLUSION: Investigation of the genotypes and clinical data in patients carrying the R143W variant on an allele indicated that severity of hearing loss depends largely on the levels of dysfunction of the pathogenic variant on the allele, whereas a patient with the homozygous R143W variant demonstrated profound hearing loss. We concluded that these hearing impairments may be due to destabilization of the protomer structure of GJB2 caused by the R143W variant.

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.

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.

[Mutation spectrum analysis of 23-site chip neonatal deafness genetic screening].

Objective:To analyze the mutation spectrum of 23-site chip newborn deafness genetic screening in Beijing, and to provide basis for genetic counseling and clinical diagnosis and treatment. Methods:The study included 21 006 babies born in Beijing from December 2022 to June 2023. All subjects underwent newborn deafness genetic screening in Beijing Tongren Hospital, covering 23 variants in 4 genes, the GJB2 gene（c.35delG, c.176_191del16, c.235delC, c.299_300delAT, c.109G>A, c.257C>G, c.512insAACG, c.427C>T, c.35insG）, SLC26A4 gene（c.919-2A>G, c.2168A>G, c.1174A>T, c.1226G>A, c.1229C>T, c.1975G>C, c.2027T>A, c.589G>A, c.1707+5G>A, c.917insG, c.281C>T）, Mt12SrRNA（m.1555A>G, m.1494C>T） and GJB3 gene（c.538C>T）. The mutation detection rate and allele frequency were analyzed. Results:The overall mutation detection rate was 11.516%（2 419/21 006）, with the GJB2 gene being the most frequently involved at 9.097%（1 911/21 006）, followed by the SLC26A4 gene at 2.123%（446/21 006）, the GJB3 gene at 0.362%（76/21 006） and Mt12SrRNA at 0.176%（37/21 006）. Among the GJB2 genes, c.109G>A and c.235delC mutation detection rates were the highest, with 6.579%（1 382/21 006） and 1.795%（377/21 006）, respectively. Of the SLC26A4 genes, c.919-2A>G and c.2168A>G had the highest mutation rates of 1.423%（299/21 006） and 0.233%（49/21 106）, respectively. Regarding the allele frequency, GJB2 c.109G>A was the most common variant with an allele frequency of 3.359%（1 411/42 012）, followed by the GJB2 c.235delC at 0.897%（377/42 012） and the SLC26A4 c.919-2A>G at 0.719%（302/42 012）. Conclusion:23-site chip newborn deafness genetic screening in Beijing showed that GJB2 c.109G>A mutation detection rate and allele frequency were the highest. This study has enriched the epidemiological data of 23-site chip genetic screening mutation profiles for neonatal deafness, which can provide evidence for clinical practice.

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.