Gap junction protein connexin-43 and its distribution in different tissues

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1. Connexins and gap junctions

1.1. Connexin family

Connexins (Cx), or gap junction proteins, are a family of transmembrane proteins that form a special type of membrane channel in the cell, the gap junction or nexus. Connexins are found only in vertebrates, whereas in invertebrates similar functions are performed by innexins, which are not homologous to connexins. There are 21 genes encoding connexins in the human genome. These genes are distributed across different chromosomes, but generally, they are located near other connexin-coding genes, forming groups of two to five genes. The connexin family is divided into five subfamilies, designated GJA, GJB, GJC, GJD, and GJE. The designation GJ (gap junction) indicates that the protein belongs to the connexin family, and the third letter indicates the subfamily. Proteins are named according to their molecular mass, e.g., connexin-26 has a molecular mass of 26 kDa and is designated Cx26. Connexin sequences exhibit high degrees of identity between paralogs and orthologs across diverse vertebrate species. In fact, orthologs in various species often exhibit identical molecular masses. All connexins share a similar structural organization, comprising four transmembrane segments, unstructured N- and C-termini facing the cytoplasm, a cytoplasmic loop, and two extracellular loops (EL1 and EL2).

1.2. Connexin-43

Cx43 is the most abundant connexin in the human body. Currently, there are more than 50 cell types expressing Cx43. It is encoded by the GJA1 gene, which is located on the sixth chromosome in humans (6q22.31), is 14,081 bp long, and consists of two exons [3]. The protein was first characterized in 1987 by Beyer et al. (1987).

An intriguing aspect of GJA1 mRNA is the presence of an IRES, which enables cap-independent translation of Cx43. This may be crucial under stress conditions when cap-dependent translation is impeded, such as during apoptosis or at specific stages of the cell cycle [5].

Cx43, as the name implies, has a mass of 43 kDa and consists of 382 amino acids. Its structure is analogous to that of other connexins. In experiments with mutant forms, the functional significance of each structural element of Cx43 was elucidated. The N-terminal domain is of paramount importance for the oligomerization of connexins and regulation of gap junction permeability. Transmembrane domains are hydrophobic and can anchor in the plasmalemma, forming the gap junction pore, and regulating heteromer compatibility. Extracellular loops regulate the proper coupling of hemicanals during gap junction assembly. The cytoplasmic loop, by binding to the C-terminal domain, is involved in pH-mediated regulation of channel permeability and contains putative binding sites for other connexins. The C-terminal domain is the most stable part of the molecule, exhibiting a high degree of conservativity. This domain is practically immune to mutations, which are lethal at the early stages of embryogenesis [6].

The C-terminal domain consists of 150 amino acids (residues 232–382), among which serine (22 residues) and proline (13 residues) constitute a significant proportion. This amino acid composition makes the C-terminus a target for multiple protein kinases, which is critical for the regulation of connexin-mediated intercellular interactions. Modeling of the secondary structure of the C-terminal domain indicates that it has a linear structure, which offers great potential for interaction with various proteins. Phosphorylation of the C-terminus affects many properties of connexin and life cycle events of the molecule. These include channel capacity, delivery, assembly, endocytosis, and degradation. MAP kinase (MAPK), protein kinase C, casein kinase 1, Src tyrosine kinase, tyrosine kinase 2, cyclin-dependent kinase 1, and presumably other kinases, such as calmodulin-dependent kinase II, are involved in regulating the life cycle of Cx43. Phosphatases that directly interact with the C-terminus of Cx43 include T-cell protein tyrosine phosphatase. In addition to phosphorylation and dephosphorylation, the C-terminal domain is susceptible to acetylation, S-nitrosylation, ubiquitination, and sumoylation. Furthermore, the C-terminus of Cx43 interacts with cytoskeleton proteins. It binds directly to tubulin and indirectly to actin through the adaptor proteins zonula occludens-1 (ZO-1) and drebrin (developmentally regulated brain protein). Another group of proteins that interacts with Cx43 includes regulators of cell growth, migration, and differentiation, such as beta-catenin, CCN3, cyclin-E, caveolin-1, Hsc70, beta-arrestin, and Вах. Integrin α5 serves as a modulator of hemicanal activity, capable of direct binding to the C-terminus [7]. These findings indicate that the C-terminal domain of the Cx43 molecule is highly important in regulating the gap junction structure and its functions at all stages of its existence.

Like other connexins, Cx43 is highly conservative. Its primary structure is highly conserved across most vertebrates [8]. Several features make Cx43 the most representative member of the family. First, it is the most widely expressed connexin in the human body and is found in cells of almost all vital organs, including the brain, heart, lung, stomach, and intestines. Second, Cx43 is most susceptible to phosphorylation by connexin, which is necessary for regulating the gap junction life cycle. Finally, as with other connexins, Cx43 is characterized by a very short half-life of 2–4 h. It is assumed that a complete change of Cx43 molecules occurs in the heart within a day [9].

1.3. Structure and properties of the gap junction

Initially, insight into the molecular organization of the gap junction was obtained by X-ray diffraction analysis of Cx26 [10], but, recently, the structure of Cx43 has been established by cryoelectron microscopy [11]. All gap junctions, including those consisting of innexins and pannexins, have a uniform structure: six connexin molecules (or other gap junction molecules) oligomerize to form a pore, the connexon. The hydrophobicity and specificity of the transmembrane domains are crucial for the assembly of the connexon [12]. In cells expressing multiple connexins, there is a high probability of co-oligomerization, which results in the formation of heteromeric connexons. However, only certain pairs of connexins can form heteromers, which is important for further functional separation of the interactomes formed by gap junctions. Extracellular loops play a key role in connecting two connexons to form gap junctions, which are also responsible for the specificity of interactions between connexons composed of various connexins. A connexon of one cell that is not connected to a connexon of another cell forms a hemicanal that allows communication between the cell and extracellular environment [13].

Connexin molecules are synthesized in the endoplasmic reticulum before they enter the Golgi apparatus. Connexons are assembled in the trans part of the Golgi apparatus, after which they are transported to the plasma membrane, where they accumulate at sites rich in N-cadherin and ZO 1. In the membrane, gap junctions (or hemichannels) form plaques, which are nexuses containing several tens to thousands of connexions, of which 10%–20% are functional. Plaques have an approximate diameter of 0.5 μm and can further aggregate into structures of larger size, the shape of which differs in various cell types. For instance, in the myocardium, nexuses are agglutinated as part of insertion disks, whereas in other cells, such as astrocytes, they do not form complex, ordered structures [14]. The assembly of gap junctions is regulated by multiple factors, with C-terminal phosphorylation being of particular significance. Channel closure, internalization, and degradation are mediated by multiple events, with phosphorylation by MAPK, PKC, Src, and CDC2 being of particular note. Their brief half-life indicate that gap junctions exist in a state of unstable equilibrium, oscillating between assembly and degradation. This equilibrium is primarily regulated by modifications to the C-terminal domain [15], which determines the lability of intercellular junctions.

2. Distribution and function of Cx43 in various tissues

2.1. Myocardial muscle

The initial identification and subsequent investigation of Сх43 commenced with the study of cardiomyocytes. Consequently, one of the earliest descriptions of this connexin was derived from the analysis of rat myocardial cells [4], and subsequent models constructed using cardiomyocytes derived from human stem cells were used in studies of intercellular communication [16]. The earliest studies of electrical conduction in the myocardium indicated that cardiomyocytes are united in a functional network, commonly referred to as a functional syncytium. Myocardial insertion disks play a key role in the formation of a syncytium. One of the main elements of a myocardial insertion disk is a nexus, which, is predominantly composed of Сх43 in the cardiac muscle [17].

The most crucial property of gap junctions that enables them to participate in the synchronization of cardiac contractions is their capacity to facilitate the transfer of Ca²⁺ between connected cells [18]. The nexus is regarded as the primary structural component of the insertion disk, a specific intercellular junction formed by cardiomyocytes. The image of Cx43 in the insertion disks of human myocardial cardiomyocytes obtained by confocal microscopy is presented in the Figure. Calcium and the metabolic connectivity of syncytium cells play a significant role in cardiomyocyte association. This is because gap junctions can allow the passage of several small molecules, including amino acids, nucleotides, adenosine triphosphate, and hormones. Thus, the term “metabolic syncytium” is also often used [19].

 

Figure. Detection of intercalated discs in human heart cardiomyocytes using the reaction of Cx43 (specimen from the archives of the Department of General and Special Morphology of the Institute of Experimental Medicine). Cardiomyocyte nuclei are stained with SYTOX Green dye, Cx43 is detected by antibodies visualized with Cy3 fluorochrome

 

Cx43 plays a pivotal role in both the study of normal cardiac physiology and the modeling of pathologies. One observed phenomenon is that susceptibility to arrhythmias increases with a decrease in Cx43 levels in myocardial cells. This phenomenon has been demonstrated in mice with induced knockout of the GJA1 gene [20] and in rats exposed to high stress levels [21]. Another observed phenomenon is a shift of gap junctions from the insertion disks to the lateral membranes of cardiomyocytes, which has been termed “lateralization” [22]. This has been observed, for example, in cardiac dysfunction in patients with lethal COVID-19 [23] and in oxidative stress [24]. The spectrum of conditions wherein there is a change in the normal expression and localization of Cx43 in the heart is quite broad and includes hypertrophic cardiomyopathy, heart failure, and ischemia [25].

The relationship between gap junctions and various pathologies allows for the use of Cx43 as a target for pharmacological therapy, for example, in heart rhythm disorders. Drugs used in this therapy are typically peptides that selectively modulate the function of connexin gap junctions or connexin mimetic peptides, which are artificially synthesized proteins with similarity to connexins [26–28].

2.2. Nerve tissue

Although it was intially regarded as a cardiac connexin, Cx43 is now investigated as the major gap junction protein of astrocytes. In addition to astrocytes, proteins from the connexin family in the central nervous system (CNS) are found in oligodendrocytes, ependymocytes, tanycytes, and neurons. However, Cx43 is specific to astrocytes, ependymocytes, and tanycytes [29]. In the CNS, gap junctions of Cx43 do not form regular, structured formations, but rather cluster into plaques with a diameter of 0.5–2.5 μm, which varies depending on the localization. Connexin contacts are located both on the cell bodies of glia and on their outgrowths, and the density of immunohistochemical staining differs in different brain structures [30].

Interestingly, the expression pattern of connexins exhibits considerable heterogeneity both between various cell types and within populations. For instance, Cx30, Cx43, and presumably Cx26 occur in astrocytes, whereas Cx29, Cx32, and Cx47 occur in oligodendrocytes. Additionally, studies of the molecular composition of gap junctions in astrocytes have corroborated the hypothesis that not all combinations of connexins can form a functional contact. Astrocytic connexins are predominantly formed from Cx30/Cx30 and Cx43/Cx43 pairs. The prevalence of gap junctions with one or another composition differs in different parts of the CNS. Cx43 is expressed earlier in ontogenesis and is widespread, whereas Cx30 is expressed later and predominantly occurs in the gray matter. Most of the gap junctions in oligodendrocytes are heterotypic, comprising pairs of various connexins, and heterologous, involving the participation of various cell types in their formation. These are formed by Cx32/Cx30 and Cx47/Cx43 pairs, respectively [33]. Recent in vitro studies indicate that the potential functional combinations may extend beyond the previously identified pairs [34]. Initially, it was assumed that oligodendrocytes lacked gap junctions, but subsequent investigations have revealed that such a connection persists, with a greater prevalence observed in the presence of homotypic Cx32 contacts [35]. To date, Cx36 is considered to be the main neuronal connexin, which does not form functional pairs with any glial connexin. This suggests a functional separation of glial and neuronal networks formed by gap junctions [36, 37].

Another crucial inquiry in CNS research pertains to the function of Cx43 in ependymal cells. Rash et al. [31], who focused on the expression of Cx43 in astrocytes, noted the presence of this protein in ependyma. They proposed that ependymocytes constitute a fully integrated component of the panglionic syncytium. Subsequent studies have highlighted the potential role of gap junctions between tanycytes and ependymocytes in synchronizing the movements of microvilli [38, 39]. Another interesting hypothesis is the potential association of Cx43-positive ependymocytes with astrocytes, particularly if there are CNS injuries. Liu et al. [40] reported the morphological similarity of GFAP-positive ependymocytes with astrocytes. Roales-Buján et al. [41] demonstrated that in a hydrocephalus model, damaged areas of ependyma are replaced by astrocytes that exhibit morphological similarities to ependymal cells. However, in both studies, Cx43 is considered a marker specific for astrocytes although its expression in normal ependyma has been repeatedly demonstrated.

In the peripheral nervous system, the presence of Cx43 has been demonstrated primarily in Schwann cells, as well as in fibroblasts of connective tissue sheaths of peripheral nerves [42, 43] and satellite cells of spinal ganglia [44]. In Schwann cells, the expression level of Cx43 is considerably lower than that of Cx32, which makes its detection challenging. Cx43 occurs in both myelin sheath layers and Schwann cell bodies. Currently, there is a paucity of information regarding the functional role of Cx43 in the peripheral nervous system. This represents a potential avenue for future research.

2.3. Testicular tissue

Another organ in which the functions of Cx43 are intensively studied is the mammalian testis. In this organ, Cx43 is expressed in the membranes of Leydig cells and Sertoli cells, and diffusely in the cytoplasm of spermatocytes and spermatogoniums. Additionally, Cx43 expression depends on the age and maturation stage of the spermatogenic epithelium [45]. In Sertoli cells, Cx43 occurs in the region of the blood–brain barrier and in the sites of contact with spermatocytes. This distribution is not only exclusive to mammals [46–50] but is also observed in reptiles [51] and amphibians [52].

Studies on GJA1 knockout mouse lines and on individuals with impaired spermatogenesis have demonstrated that loss of fertility can be associated with low expression of Cx43 [53–55], with disruption of its spatial distribution [56] or caused by artificial blocking of connexin channels [57]. Spermatogenesis arrest occurred at the level of spermatogoniums in GJA1 knockout mice.

These observations raised the question of the role of Cx43 in the testes. It is assumed that Cx43 in Sertoli cells forms a functional syncytium that allows synchronizing the metabolic activity of these cells during spermatogenesis [58]. Other functions of connexin include the regulation of proliferation, maturation, and apoptosis of Sertoli cells [57, 59, 60], synchronization of androgenic activity of Leydig cells with the stages of spermatogenesis [61], regulation of germline cell migration to the testes and their survival [62], and control of the dynamics and composition of the hematotesticular barrier [63]. The role of Cx43 in the pathogenesis of testicular tumors is currently under investigation. Its potential as a prognostic and diagnostic marker in the development of testicular tumors has been suggested [64].

2.4. Lymphoid tissue

After the isolation of Cx43 from cardiomyocytes, it was demonstrated that this specific connexin forms gap junctions in epitheliocytes of the brain matter of the thymus and in the stroma of human and mouse bone marrow [65, 66]. As in all tissues previously considered, Cx43 forms a functional syncytium that coordinates lymphopoietic activity in these organs [67]. A comparable pattern was observed in secondary lymphoid organs, wherein gap junctions regulate the formation of germinal centers within follicles of lymph nodes [68].

The study of the role of Cx43 in lymphopoiesis has been hindered by the fact that fully GJA1 knockout mice die within hours after birth Ishikawa et al. (2012) demonstrated that one of the functions of Cx43 in hematopoiesis is participation in the removal of reactive oxygen species from stem cells in mice with induced GJA1 knockout [69].

Several previous studies have demonstrated that Cx43 is the primary gap junction protein in maturing leukocytes and is involved in numerous immune interactions. Cx43 is the sole connexin found in lymphoid tissue [70], which suggests its pivotal role in the processes of lymphocyte differentiation in the thymus and germinal centers of lymph nodes.

2.5. Other tissues

The initial studies of Cx43 distribution in various tissues revealed that it occurs in almost all internal organs. This section will discuss the peculiarities of Cx43 distribution and function in those tissues where it is not the only or main connexin or where its functions have not been sufficiently investigated.

Gap junctions occur in all types of mammalian epithelia. However, the distribution of certain connexins may differ in different epithelial tissues. In humans, Cx43 is the most prevalent connexin in epithelia, with a broad distribution throughout various organs and tissues. It is found in the oral cavity, esophagus, and stomach, as well as in myoepithelial cells of salivary glands, epithelium of testes, cells of renal tubules and tubule, epithelial cells of lungs and thyroid gland, and other locations [2, 71]. The distribution of Cx43 in the epidermis has been previously studied. It is abundantly expressed in keratinocytes and, accordingly, can be found in all layers of multilayer epithelium [72, 73]. In many pathological conditions, the content of Cx43 is altered. For example, its level increases in chronic eczema and psoriasis, and decreases in acute eczema [74].

Extensive research is being conducted to elucidate the structure and functions of Cx43 in blood vessels. Cx43 occurs in vascular smooth muscle, endothelium, and pericytes [75]. Although Cx43 expression varies with vessel size, location, species, and cell type, it plays a pivotal role in maintaining blood vessel function in normal blood vessels. Cx43 is involved in the formation of heterotypic and homotypic contacts between smooth muscle, endothelial cells, and pericytes. Cx43 plays a role in maintaining vascular tone, proliferation, angiogenesis, and facilitating endothelial cell barrier function. Alterations in the expression, localization, and posttranslational modification of Cx43 are frequently observed in vascular diseases, such as atherosclerosis, hypertension, and diabetes mellitus [76].

Apart from vascular smooth muscle, Cx43 occurs in most smooth muscle cells throughout the body. Of particular interest in this regard are the muscular layers of the intestinal, bladder, and uterine sheaths. The ability of connexin to conduct Ca²⁺ signaling, linking cells into a single functional network, appears to be important for the rhythmic contractions of the muscular lining of large hollow organs. As in the case of myocardium, the connection of smooth muscle cells by gap junctions is critical for the synchronization of muscle contractions, especially under conditions where only groups of cells rather than individual myocytes are innervated.

Another tissue type wherein the distribution of Cx43 is not fully understood is adipose tissue. In mammals, there are two types of adipose tissue: white and brown. White adipose tissue is the dominant type and performs a multitude of functions, including structural, metabolic, homeostatic, and endocrine functions. Brown adipose tissue is renowned for its capacity to generate heat through energy expenditure [80].

Cx43 has been the subject of the most comprehensive research in brown adipose tissue, where it plays a pivotal role in adipogenesis and the maintenance of tissue homeostasis [81, 82]. It has been assumed that the presence of Cx43 was not characteristic of white adipose tissue. However, recent studies have demonstrated a role for this protein analogous to that in brown adipose tissue, suggesting the importance of Cx43 in the secretory activity of white adipose tissue [83, 84]. The observation that the amount of Cx43 decreases in adipose tissue during adipogenesis, and overexpression blocks this process, has led to the conclusion that the regulation of Cx43 is linked to obesity in humans [85].

Cx32 is the principal hepatic connexin expressed in hepatocytes and endothelial cells of hepatic sinusoids[86], whereas, Cx43 occurs in Kupffer cells, perfused perisinusoidal cells, glisson capsule mesothelial cells, and cholangiocytes [87–90]. The role of Cx43 in the liver is unclear. Most probably, it is involved in the regulation of proliferation, differentiation, and death of hepatocytes [91].

A further area of interest is the study of Cx43’s involvement in the development of cancer and prediction of chemotherapy efficacy. This protein plays an important role in chemotherapy and “suicidal gene” therapy. Some antitumor drugs are able to diffuse into the tumor cell population through gap junctions, which may enhance their therapeutic effect [92–98]. Additionally, a direct role of Cx43 in tumor diseases has been demonstrated [99].

Conclusions

Many conclusions can be drawn from the reviewed literature on Cx43. It appears that in mammals, one of the key structural elements of histohematic barriers formed between blood and tissues is subjected to autoimmune destruction. The barrier functionality of Cx43 is most pronounced in the formation of the blood–brain barrier and hematotesticular barrier. In addition to histohematic barriers, connexion also typically occurs in blood-brain barriers. Cx43 occurs in all typical epithelial tissues. A structurally similar pattern is observed in both endothelium and ependyma, despite their structural and histogenetic differences from typical epithelia.

Another specific function of Cx43 is functional synchronization of large cell populations, especially under circumstances where direct innervation or blood supply may be difficult. In such instances, the functional syncytium ensures synchronized and uniform action of its constituent cells. This phenomenon is most evident in the myocardium, smooth muscle of blood vessels, and brain astrocytes. The functional connection of cells of such tissues, provided by connexins, is necessary for their coordinated work. Its disruption inevitably leads to serious consequences, often lethal. Nevertheless, to date, much about Cx43 remains unknown. For example, the mechanisms by which Cx43 is linked to proliferative processes are still unknown. The role of Cx43 in the structures of the peripheral nervous system is also still unclear.

Additional information

Funding source. The study was financially supported by the Institute of Experimental Medicine.

Ethical approval. As part of the study, work on biological objects was not carried out.

Conflict of interest. The authors declare no conflict of interest.

Authors̕ contribution. All authors made significant contributions to concept development, research and paper preparation, read and approved the final version before publication. The largest contribution is distributed as follows: M.S. Filippov — literature search and review, text writing; D.Е. Korzhevskii — article structure design, text editing.

About the authors

Mikhail S. Filippov

Institute of Experimental Medicine

Email: msfilippov@mail.ru
SPIN-code: 7789-7219

Research Laboratory Assistant, Laboratory of Functional Morphology of the Central and Peripheral Nervous System, Department of General and Special Morphology

Russian Federation, Saint Petersburg

Dmitrii E. Korzhevskii

Institute of Experimental Medicine

Author for correspondence.
Email: DEK2@yandex.ru
ORCID iD: 0000-0002-2456-8165
SPIN-code: 3252-3029

MD, Dr. Sci. (Med.), Professor of the Russian Academy of Sciences, Head of the Laboratory of Functional Morphology of the Central and Peripheral Nervous System, Department of General and Special Morphology

Russian Federation, Saint Petersburg

References

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