Connexin Gene Family Research Articles

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and invivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.

Endocytosis and Endocytic Motifs across the Connexin Gene Family.

Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.

Multi-omics analysis of expression profile and prognostic values of connexin family in LUAD.

Our study first explored the expression differences and prognostic significance of Cx genes in pan-cancer and then focused on LUAD. Our objectives were to conducted a comprehensive analysis of the expression profile, prognostic significance, genetic alterations, potential biological functions and drug sensitivity of the Connexin gene family in LUAD. We developed a comprehensive prognostic model for LUAD by combining risk scores with clinical features and created a nomogram to predict 1-, 3-, and 5-year overall survival. Using single-cell sequencing, we examined the expression and biological functions of the identified prognostic markers. Our risk model revealed that GJB2-5 play a critical role in the prognosis of LUAD patients, associated with many biological processes such as cell cycle, DNA damage, EMT, hypoxia, invasion, and metastasis. Furthermore, the connexin gene family is linked to transcriptional mechanisms such as the extracellular matrix (ECM), migration, mobility, angiogenesis, and the epithelial-mesenchymal transition (EMT) genetic program. The risk model can be used as a potential prognostic factor for LUAD patients and may provide new insights into cancer treatment from perspective of the expression of Cx genes.

Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Genome-wide association study of varicose veins identifies a protective missense variant in GJD3 enriched in the Finnish population

Varicose veins is the most common manifestation of chronic venous disease that displays female-biased incidence. To identify protein-inactivating variants that could guide identification of drug target genes for varicose veins and genetic evidence for the disease prevalence difference between the sexes, we conducted a genome-wide association study of varicose veins in Finns using the FinnGen dataset with 17,027 cases and 190,028 controls. We identified 50 associated genetic loci (P < 5.0 × 10−8) of which 29 were novel including one near ERG with female-specificity (rs2836405-G, OR[95% CI] = 1.09[1.05–1.13], P = 3.1 × 10−8). These also include two X-chromosomal (ARHGAP6 and SRPX) and two autosomal novel loci (TGFB2 and GJD3) with protein-coding lead variants enriched above 56-fold in Finns over non-Finnish non-Estonian Europeans. A low-frequency missense variant in GJD3 (p.Pro59Thr) is exclusively associated with a lower risk for varicose veins (OR = 0.62 [0.55–0.70], P = 1.0 × 10−14) in a phenome-wide scan of the FinnGen data. The absence of observed pleiotropy and its membership of the connexin gene family underlines GJD3 as a potential connexin-modulating therapeutic strategy for varicose veins. Our results provide insights into varicose veins etiopathology and highlight the power of isolated populations, including Finns, to discover genetic variants that inform therapeutic development.

Connexinplexity: the spatial and temporal expression of connexin genes during vertebrate organogenesis.

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is widely used in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type-specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we determined the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step toward understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.

Gap Junction-Dependent and -Independent Functions of Connexin43 in Biology.

Simple SummaryConnexin43 is one of the 21 members of a large protein family that forms intercellular gap junction complexes. It plays a critical role in development by allowing two adjacent cells to exchange cellular content. Mouse deletion studies have demonstrated its critical roles in many organs, including cardiac muscle, blood vessels, bone, adipose tissue and more. However, as the most expressed and most studied connexin in the family, Connexin43 surprisingly shows many gap-junction-independent roles, spanning from the forming of hemichannels, to regulating membrane trafficking, to regulating mitochondrial function. Connexin43 is also unique in the family by forming many smaller polypeptides through alternative utilization of its mRNA molecule or cleavage of the translated full-length protein. This review summarizes recent advances on Connexin43’s gap-junction-dependent and -independent functions in physiology. The knowledge will expand our understanding of how a gene grows its function by multiplexing its mRNA and protein. In the end, this may also guide us to develop Connexin43-based therapy for metabolic conditions, cancer, and other related diseases. For the first time in animal evolution, the emergence of gap junctions allowed direct exchanges of cellular substances for communication between two cells. Innexin proteins constituted primordial gap junctions until the connexin protein emerged in deuterostomes and took over the gap junction function. After hundreds of millions of years of gene duplication, the connexin gene family now comprises 21 members in the human genome. Notably, GJA1, which encodes the Connexin43 protein, is one of the most widely expressed and commonly studied connexin genes. The loss of Gja1 in mice leads to swelling and a blockage of the right ventricular outflow tract and death of the embryos at birth, suggesting a vital role of Connexin43 gap junction in heart development. Since then, the importance of Connexin43-mediated gap junction function has been constantly expanded to other types of cells. Other than forming gap junctions, Connexin43 can also form hemichannels to release or uptake small molecules from the environment or even mediate many physiological processes in a gap junction-independent manner on plasma membranes. Surprisingly, Connexin43 also localizes to mitochondria in the cell, playing important roles in mitochondrial potassium import and respiration. At the molecular level, Connexin43 mRNA and protein are processed with very distinct mechanisms to yield carboxyl-terminal fragments with different sizes, which have their unique subcellular localization and distinct biological activities. Due to many exciting advancements in Connexin43 research, this review aims to start with a brief introduction of Connexin43 and then focuses on updating our knowledge of its gap junction-independent functions.

Cellular mechanisms of connexin-based inherited diseases.

The 21-member connexin gene family exhibits distinct tissue expression patterns that can cause a diverse array of over 30 inherited connexin-linked diseases ranging from deafness to skin defects and blindness. Intriguingly, germline mutations can cause disease in one tissue while other tissues that abundantly express the mutant connexin remain disease free, highlighting the importance of the cellular context of mutant expression. Modeling connexin pathologies in genetically modified mice and tissue-relevant cells has informed extensively on no less than a dozen gain- and loss-of-function mechanisms that underpin disease. This review focuses on how a deeper molecular understanding of the over 930 mutations in 11 connexin-encoding genes is foundational for creating a framework for therapeutic interventions.

Connexins during 500 Million Years-From Cyclostomes to Mammals.

It was previously shown that the connexin gene family had relatively similar subfamily structures in several vertebrate groups. Still, many details were left unclear. There are essentially no data between tunicates, which have connexins that cannot be divided into the classic subfamilies, and teleosts, where the subfamilies are easily recognized. There are also relatively few data for the groups that diverged between the teleosts and mammals. As many of the previously analyzed genomes have been improved, and many more genomes are available, we reanalyzed the connexin gene family and included species from all major vertebrate groups. The major results can be summarized as follows: (i) The same connexin subfamily structures are found in all Gnathostomata (jawed vertebrates), with some variations due to genome duplications, gene duplications and gene losses. (ii) In contrast to previous findings, birds do not have a lower number of connexins than other tetrapods. (iii) The cyclostomes (lampreys and hagfishes) possess genes in the alpha, beta, gamma and delta subfamilies, but only some of the genes show a phylogenetic affinity to specific genes in jawed vertebrates. Thus, two major evolutionary transformations have occurred in this gene family, from tunicates to cyclostomes and from cyclostomes to jawed vertebrates.

Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed

Connexin proteins facilitate transmembrane and intercellular communication via channel formation (hemichannel) and intercellular docking of apposed hemichannels to form gap junction channels, respectively. Additionally, they play a role in determining growth phenotype (e.g., arrest, proliferation, death). Connexin 37 (Cx37), expressed in connexin‐deficient rat insulinoma (Rin) cells induces growth arrest or cell death in a manner dependent on the phosphorylation of specific carboxyl terminus (CT) residues. Notably, phosphorylation at serine 319 (S319) is both necessary and sufficient for growth arrest, and mimicking phosphorylation with aspartate substitution at either S275 or S321 is sufficient to induce cell death. 13 of the 21 members of the connexin gene family have a conserved methionine at position 213; in Cx43 mRNA, this methionine codon serves as an alternate translation start site that produces a CT‐spanning translation product. This 20kDa (20k) product interacts with Cx43, chaperoning its trafficking to the membrane. Here we test the hypotheses that 1) this conserved codon in Cx37 mRNA also serves as an alternate translation start site to produce a 13kDa product (13k) that, when expressed alone, regulates growth phenotype of Rin cells and 2) when co‐expressed with Cx37, regulates its channel function. To test these hypotheses, we expressed V5‐tagged 13k or one of two phosphomimetic mutants, 13k‐S321D and 13k‐S275D, in Rin cells or in Rin cells expressing Cx37. Despite a similar phosphorylation profile to Cx37, as determined by mass spectrometry, 13k did not arrest Rin cell growth and neither phosphomimetic mutant induced cell death nor growth arrest. In co‐expressing cells, immunoprecipitation (IP) of 13k failed to co‐IP full‐length Cx37, suggesting no direct interaction between these isoforms; however, 13k and the phosphomimetic mutants altered Cx37 hemichannel conductance and open probability properties, suggesting an indirect interaction of 13k isoforms with Cx37. In conclusion, these data suggest that while 13k and phosphomimetic mutants alone are insufficient to alter the growth phenotype of Rin cells toward growth arrest or death, they nevertheless lead to altered channel properties of full‐length Cx37. The findings herein will inform further research in understanding how connexins are regulated and, in turn, the role they play in regulating cell growth phenotypes.Support or Funding InformationR01 HL131712 | Undergraduate Biology Research Program | University Funds

Phylogeny of teleost connexins reveals highly inconsistent intra- and interspecies use of nomenclature and misassemblies in recent teleost chromosome assemblies

BackgroundBased on an initial collecting of database sequences from the gap junction protein gene family (also called connexin genes) in a few teleosts, the naming of these sequences appeared variable. The reasons could be (i) that the structure in this family is variable across teleosts, or (ii) unfortunate naming. Rather clear rules for the naming of genes in fish and mammals have been outlined by nomenclature committees, including the naming of orthologous and ohnologous genes. We therefore analyzed the connexin gene family in teleosts in more detail. We covered the range of divergence times in teleosts (eel, Atlantic herring, zebrafish, Atlantic cod, three-spined stickleback, Japanese pufferfish and spotted pufferfish; listed from early divergence to late divergence).ResultsThe gene family pattern of connexin genes is similar across the analyzed teleosts. However, (i) several nomenclature systems are used, (ii) specific orthologous groups contain genes that are named differently in different species, (iii) several distinct genes have the same name in a species, and (iv) some genes have incorrect names. The latter includes a human connexin pseudogene, claimed as GJA4P, but which in reality is Cx39.2P (a delta subfamily gene often called GJD2like). We point out the ohnologous pairs of genes in teleosts, and we suggest a more consistent nomenclature following the outlined rules from the nomenclature committees. We further show that connexin sequences can indicate some errors in two high-quality chromosome assemblies that became available very recently.ConclusionsMinimal consistency exists in the present practice of naming teleost connexin genes. A consistent and unified nomenclature would be an advantage for future automatic annotations and would make various types of subsequent genetic analyses easier. Additionally, roughly 5% of the connexin sequences point out misassemblies in the new high-quality chromosome assemblies from herring and cod.

Connexin 43 Mutations Lead to Increased Hemichannel Functionality in Skin Disease.

Gap junctional channels are specialized components of the cellular membrane that allow the intercellular passage of small metabolites, ions, and second messengers to maintain homeostasis. They are comprised of members of the connexin gene family that encode a wide array of proteins that are expressed in nearly every tissue type. Cx43 is perceived to be the most broadly expressed connexin in humans, with several genetic skin diseases being linked to Cx43 mutations specifically. These mutations, in large, produce a gain of functional hemichannels that contribute to the phenotypes of Erythrokeratoderma Variabilis et Progressiva (EKVP), Palmoplantar Keratodemra Congenital Alopecia-1 (PPKCA1), and others that produce large conductance and increased permselectivity in otherwise quiescent structures. Gaining functional hemichannels can have adverse effects in the skin, inducing apoptosis via Ca2+ overload or increased ATP permeability. Here, we review the link between Cx43 and skin disease. We aim to provide insight into the mechanisms regulating the normal and pathophysiological gating of these essential proteins, as well as address current therapeutic strategies. We also demonstrate that transient transfection of neuro-2a (N2a) cells with mutant Cx43 cDNA resulted in increased hemichannel activity compared to wild-type Cx43 and untransfected cells, which is consistent with other studies in the current literature.

Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease.

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease.

The role of gap junctions in disease and potential treatment approaches

Our research program is engaged in examining the over two dozen distinct human diseases linked to genes that encode the proteins (connexins) used in gap junctional intercellular communication. Mutations in 50% of the 21 connexin gene family members result in conditions ranging from developmental abnormalities that include hearing loss to life-shortening organ failure. We use a multidimensional approach involving organotypic cultures, genetically-modified mice, connexinlinked disease patient cells and human inducible pluripotent stem cells to interrogate the scope of mechanisms that lead to disease in some tissues while other organs are spared. In principal, connexins are linked to disease or injury repair through three different mechanistic paradigms.

Cellular and Deafness Mechanisms Underlying Connexin Mutation-Induced Hearing Loss - A Common Hereditary Deafness.

Hearing loss due to mutations in the connexin gene family, which encodes gap junctional proteins, is a common form of hereditary deafness. In particular, connexin 26 (Cx26, GJB2) mutations are responsible for ~50% of non-syndromic hearing loss, which is the highest incidence of genetic disease. In the clinic, Cx26 mutations cause various auditory phenotypes ranging from profound congenital deafness at birth to mild, progressive hearing loss in late childhood. Recent experiments demonstrate that congenital deafness mainly results from cochlear developmental disorders rather than hair cell degeneration and endocochlear potential reduction, while late-onset hearing loss results from reduction of active cochlear amplification, even though cochlear hair cells have no connexin expression. However, there is no apparent, demonstrable relationship between specific changes in connexin (channel) functions and the phenotypes of mutation-induced hearing loss. Moreover, new experiments further demonstrate that the hypothesized K+-recycling disruption is not a principal deafness mechanism for connexin deficiency induced hearing loss. Cx30 (GJB6), Cx29 (GJC3), Cx31 (GJB3), and Cx43 (GJA1) mutations can also cause hearing loss with distinct pathological changes in the cochlea. These new studies provide invaluable information about deafness mechanisms underlying connexin mutation-induced hearing loss and also provide important information for developing new protective and therapeutic strategies for this common deafness. However, the detailed cellular mechanisms underlying these pathological changes remain unclear. Also, little is known about specific mutation-induced pathological changes in vivo and little information is available for humans. Such further studies are urgently required.

Voltage regulation of connexin channel conductance.

Voltage is an important parameter that regulates the conductance of both intercellular and plasma membrane channels (undocked hemichannels) formed by the 21 members of the mammalian connexin gene family. Connexin channels display two forms of voltage-dependence, rectification of ionic currents and voltage-dependent gating. Ionic rectification results either from asymmetries in the distribution of fixed charges due to heterotypic pairing of different hemichannels, or by channel block, arising from differences in the concentrations of divalent cations on opposite sides of the junctional plaque. This rectification likely underpins the electrical rectification observed in some electrical synapses. Both intercellular and undocked hemichannels also display two distinct forms of voltage-dependent gating, termed Vj (fast)-gating and loop (slow)-gating. This review summarizes our current understanding of the molecular determinants and mechanisms underlying these conformational changes derived from experimental, molecular-genetic, structural, and computational approaches.

Mechanisms linking connexin mutations to human diseases.

Ubiquitously expressed connexins are tetra-spanning transmembrane proteins that form intercellular gap junction channels or cell surface hemichannels. Connexins share similar topology but no sequence homology with mammalian pannexins and CALHM1 (calcium homeostasis modulator 1), which are also large-pore transmembrane channels. Of these three channel types, clinical evidence and gene sequence analysis to date have revealed that inherited human diseases are only associated with mutations in the connexin gene family. Connexin-linked diseases often present at birth or early in life and range from mild developmental abnormalities to severe organ failure such as hearing loss. Inherited connexin gene mutations can manifest as a disease by causing anomalies or defects in connexin oligomerization, folding, ability to pass quality control mechanisms or unexpected gain- or loss-of-function. This review provides examples of the way that various connexin gene mutations can cause disease via a wide range of molecular mechanisms. We also reflect on exciting strategies being explored in the connexin field and beyond with a view of translating their findings into potential connexin-disease therapeutics.

Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially "unzip" and be internalized/endocytosed into the cell that produced each connexin.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Voltage-dependent gating of the Cx32*43E1 hemichannel: Conformational changes at the channel entrances

Voltage is an important parameter that regulates the open probability of both intercellular channels (gap junctions) and undocked hemichannels formed by members of the connexin gene family. All connexin channels display two distinct voltage-gating processes, termed loop- or slow-gating and Vj- or fast-gating, which are intrinsic hemichannel properties. Previous studies have established that the loop-gate permeability barrier is formed by a large conformational change that reduces pore diameter in a region of the channel pore located at the border of the first transmembrane domain and first extracellular loop (TM1/E1), the parahelix (residues 42–51). Here, we use cadmium metal bridge formation to measure conformational changes reported by substituted cysteines at loci demarcating the intracellular (E109 and L108) and extracellular (Q56) entrance of hemichannels formed by the Cx32 chimera (Cx32*43E1). The results indicate that the intracellular pore entrance narrows from ∼15 Å to ∼10 Å with loop-gate but not apparently with Vj-gate closure. The extracellular entrance does not appear to undergo large conformational changes with either voltage-gating process. The results presented here combined with previous studies suggest that the loop-gate permeability is essentially focal, in that conformational changes in the parahelix but not the intracellular entrance are sufficient to prevent ion flux.