Connexin 43 Connects the Cumulus Cell Network Around the Oocyte
1. Introduction
Connexin 43 (Cx43) is encoded by the gene GJA1, is the most commonly expressed connexin, and is present in most cell types. Cx43 participates in numerous cellular processes, including cardiac contraction, electrical coupling, cell differentiation, and cell death (Lee et al., 2023). The six monomers of a single type of connexin assemble to create a single hemi-channel or "connexon." When two connexons from adjacent cells dock, they create an intercellular channel that then aggregates into a gap junction plaque (Fournier et al., 2025). Ions, metabolites, and second messengers move directly from one cytoplasm to another through these channels without first entering the extracellular space. Cx43 differs from many proteins because it is not an enzyme, hormone, growth factor, or soluble recognition molecule. Instead, it is a four-transmembrane spanning protein whose primary product is a channel. Beyond forming intercellular connections, free hemi-channels can be activated under pathological conditions, and the protein also exists within mitochondria; a smaller fragment produced via alternative splicing of the Cx43 mRNA plays important roles in both trafficking and mitochondrial regulation (Boengler et al., 2022).
Because of its role in reproduction, animal scientists are interested in Cx43. Granulosa cells are primarily connected via Cx43, while connexin 37 (Cx37) is the major connexin forming connections between the oocyte surface and surrounding cells (Turathum et al., 2021). In addition, studies have localized Cx43 to both the oolemma and the transzonal projections of porcine oocytes during in vitro maturation (Lee et al., 2016). This review examines Cx43 from a protein-centered perspective, covering its structure, life cycle, channel modes, isoforms, mitochondrial functions, roles in the cumulus–oocyte complex and porcine IVM, disease associations, and therapeutic targeting.
2. Structural Organization of Connexin 43
Human Cx43 contains 382 amino acids (Lee et al., 2023). Each human Cx43 monomer has four transmembrane helices (TM1-TM4), two extracellular loops (ECL1-ECL2), a single cytoplasmic loop, and both cytoplasmic N- and C-termini. Docking between adjacent cell membranes' hemichannels is mediated by the extracellular loops. Residues 232-382 make up most of the sequence not conserved across connexin proteins and serve as the primary site for post-translational modifications (Pun et al., 2023). Cryo-electron microscopy (Cryo-EM) in 2023 enabled the first high-resolution reconstruction of the human Cx43 gap junction channel. A dodecamer formed by two hexamers of the hemichannel. The N-terminal domain lies parallel to the membrane surface; TM2 is positioned away from the pore center, leaving six peripheral openings and one large central opening. Sterol-like densities appear in the pore and between each pair of N-terminal domains. Molecular dynamics simulations show that these molecules seal the channel, suggesting this structure represents a possible closed state. The cytoplasmic loop and the C-terminal tail were not resolved (Qi et al., 2023).
In addition to providing a dodecameric representation of the channel structure, a second cryo-EM study examined how environmental and mutational changes affected its conformation. This work provided evidence for three forms of the N-terminal alpha-helix (termed gate-covering, pore-lining and flexible intermediate) based on structures determined using cryo-EM. These studies also showed that cholesteryl hemisuccinate shifted the equilibrium to favor an increase in the gate-covering form. In contrast, mutations to the C-terminal region or altered pH shift the equilibrium to favor the pore-lining form. For some of these states, an alpha-to-pi transition in TM1 opens up a secondary pathway into the membrane space (Lee et al., 2023). Figure 1 shows the dodecameric channel and the position of a single monomer within it. Panels A and B show the cryo-EM density map and model of the dodecameric channel at 2.26 Å, with each monomer colored; panel C shows one monomer within the channel. Panel D (alignment with Cx26, Cx46 and Cx31.3).
Figure 1. Cryo-EM structure of the human Cx43 gap junction channel and the position of a single Cx43 monomer.
3. Life Cycle, Turnover and Phosphorylation
Cx43 is synthesized in the endoplasmic reticulum, oligomerized into hemichannels in the Golgi apparatus and delivered to the plasma membrane as closed hemichannels (Boengler et al., 2022). Delivery to cell borders depends in part on GJA1-20k, a short internally translated isoform that acts as a trafficking partner for the full-length protein (Fournier et al., 2025). Once in the membrane, undocked hemichannels collect in the perinexus, the zone surrounding the gap junction plaque. The plaque is renewed continuously. Each gap junction has a half-life of only a few hours, after which internalized connexons are degraded through proteasomal or lysosomal pathways. The C-terminal tail controls much of this cycle. It is phosphorylated, acetylated, S-nitrosylated, ubiquitinated and SUMOylated, and its serine and tyrosine residues are targeted by Src, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC). PKC isozymes phosphorylate serine 368 (S368), which lowers channel permeability and promotes internalization and degradation. PKCγ-driven disassembly also requires zonula occludens-1 (ZO-1), which binds the C-terminal tail near S368 (Pun et al., 2023).
4. Gap Junction Channels and Hemichannels
Cx43 operates in two channel types. Open hemichannels function as part of gap junctions, while closed free hemichannels rest in the plasma membrane. During metabolic blockade or cardiac ischemia, free hemichannels can be opened. Opening of free hemichannels in cardiomyocytes during diastole allows sodium and calcium ions to enter the cell via these channels, each with a single-channel conductivity of approximately 220 pS. This increases membrane depolarization, disturbing normal calcium ion signaling within the cell (Boengler et al., 2022).
Hemichannel clusters near the perinexus area in the heart may serve structural roles. In this zone, Cx43 hemichannel clusters lie near Nav1.5 sodium channels. Ephaptic coupling has been suggested between such adjacent membranes, where tight apposition is required for electrical conduction. Thus, Cx43 participates in tissue function through both docked channels and free hemichannel openings, as well as the membrane zones surrounding these openings.
5. Internally Translated Isoforms and GJA1-20k
The GJA1 messenger RNA produces more than one protein. Internal translation from downstream methionines generates six N-terminally truncated isoforms, named by molecular weight as GJA1-32k, GJA1-29k, GJA1-26k, GJA1-20k, GJA1-11k and GJA1-7k. GJA1-20k starts at methionine 213 and is usually the most abundant. It contains part of TM4 and the entire C-terminal tail, so it cannot form a hemichannel, but it retains the tail's regulatory features. Cardiac studies first linked GJA1-20k to delivery of full-length Cx43 to the plasma membrane, cytoskeletal dynamics, and mitochondrial fission and distribution. GJA1-20k is also expressed in cancer cells, where it may contribute to epithelial-to-mesenchymal transition and metastatic behavior (Fournier et al., 2025).
6. Mitochondrial Connexin 43
Cx43 is also located within mitochondria, primarily found in the subsarcolemmal mitochondria of ventricular cardiomyocytes. Import into the mitochondria occurs via a pathway that uses the chaperone Hsp90 and the outer-membrane translocase (TOM), and most data indicate it resides at the inner mitochondrial membrane. GJA1-20k is localized at the outer membrane of the mitochondria. Mitochondrial Cx43 appears to allow potassium ions to enter, affecting respiration and reactive oxygen species production. Both ischemic and diazoxide preconditioning use mitochondrial Cx43 to some extent; however, its role in post-conditioning is poorly understood. The estimated conductance of mitochondrial Cx43 hemichannels is about 130 pS, lower than that of plasma membrane hemichannels (Boengler et al., 2022). GJA1-20k provides an additional level of mitochondrial organization, as it has been linked to mitochondrial fission and subcellular localization (Fournier et al., 2025). Thus, these two forms affect mitochondria differently. Figure 2 summarizes the import route, membrane location, and proposed functions of mitochondrial connexins. It shows the Hsp90/TOM import pathway, Cx43's effects on oxidative phosphorylation, potassium entry, and reactive oxygen species, and the outer-membrane location of Gja1-20k.
Figure 2. Localization, import and proposed functions of mitochondrial Cx43, including the outer-membrane position of GJA1-20k.
7. Connexin 43 in the Cumulus–Oocyte Complex
Cumulus cells reach the oocyte through transzonal projections (TZPs), thin cytoplasmic extensions that cross the zona pellucida and form gap junctions at their tips. Three-dimensional electron microscopy of mouse follicles showed that most TZPs do not reach the oocyte. Each cumulus cell carried, on average, 31 ± 5 free-ended TZPs and 9 ± 2 connected TZPs, and 78% of TZP contacts were between projections from different cells, some of which were confirmed as gap junctions (Baena and Terasaki, 2019). The TZP network is therefore also a network of cumulus-to-cumulus contacts. The two main follicular connexins are divided as follows: Cx43 is found mainly in granulosa and cumulus cells. It is thought to mediate communication between cumulus cells, whereas Cx37 is found in the oocyte and cumulus cells and is thought to mediate cumulus-to-oocyte communication. In mice lacking Cx37, Cx43 expressed in the oocyte restored cumulus–oocyte complex formation and fertility, although Cx37 channels differ in their selectivity for larger molecules and ions (Xie et al., 2023).
Gonadotropins regulate Cx43 in opposite directions. Follicle-stimulating hormone (FSH) increases Cx43 expression and gap junction formation. Luteinizing hormone (LH) activates MAPK-dependent phosphorylation of Cx43, which closes the junctions and reduces the protein; in bovine cumulus cells, Cx43 messenger RNA falls about six hours after the LH surge. Gap junctions control the movement of cyclic GMP (cGMP) and cyclic AMP (cAMP) into the oocyte, and the fall in oocyte cGMP after junction closure allows cAMP hydrolysis and the resumption of meiosis (Kordowitzki et al., 2021). Upstream of these events, LH acts on mural granulosa cells to release epidermal growth factor (EGF) receptor ligands, which activate ERK/MAPK signaling in cumulus cells and drive actin reorganization and TZP retraction (Turathum et al., 2021). Figure 3 summarizes how FSH and LH regulate Cx43 and Cx37 channels before ovulation and during meiotic resumption. Panel A shows FSH-driven channel synthesis and LH-driven disruption in the antral follicle; panel B shows LH-induced Cx43 phosphorylation, Cx37 closure and the cAMP and maturation-promoting factor changes that lead to germinal vesicle breakdown.
Figure 3. Regulation of Cx43- and Cx37-based gap junction communication in the antral follicle before ovulation and during meiotic resumption.
8. Connexin 43 in Porcine Oocyte Maturation
Porcine cumulus-oocyte complexes have been widely used to study Cx43. Most research to date has focused on these complexes as an integrated system rather than on individual components. Maturing porcine complexes in NCSU23 medium and analyzing them at 0, 14, 28, and 42 hours revealed that the Cx43 signal was present on the oolemma, TZPs, and the surface of the zona pellucida. Following maturation, Cx43 band density initially increased until 14 hours post-maturation, after which it began to decline (Lee et al., 2016). The observed changes in Cx43 band density correlate well with expected changes in functional connectivity as the oocyte resumes meiotic progression. In addition to disrupting communication between cumulus cells and the oocyte, FSH stimulates the release of EGF-like factors, including amphiregulin (AREG) and epiregulin (EREG), which signal from granulosa cells through cumulus cells to the oocyte (Turathum et al., 2021). Therefore, in porcine IVM systems, gonadotropin supplementation directly influences the pathway(s) that regulate Cx43. Culturing porcine cumulus-oocyte complexes under conditions of stress can alter cumulus cell-oocyte interactions. Heat exposure in porcine IVM cultures significantly reduced the percentage of oocytes reaching MII by 44 hours (from 77% to 6%), along with reductions in TZP numbers and TZP F-actin content. mRNA levels of β-actin and γ-actin also decreased in cumulus cells, and colocalization between TZPs and gap junctions was lower than when identified using antibodies against connexin 45 (Yin et al., 2020). To date, few studies have measured the direct effects of mycotoxin exposure (e.g., deoxynivalenol) on Cx43 localization or TZP density in porcine complexes; however, cumulus expansion and nuclear maturation depend on effective signaling between cumulus cells.
9. Connexin 43 in Disease
Mutations in GJA1 or misregulation of Cx43 cause or contribute to oculodentodigital dysplasia, palmoplantar keratoderma, heart disease and cancer (Lee et al., 2023). In the heart, the direction of change depends on the condition. S368 phosphorylation increases in some models of ischemia–reperfusion, myocarditis, and arrhythmia, but decreases in furazolidone-induced dilated cardiomyopathy, plakophilin-2-knockout arrhythmogenic right ventricular cardiomyopathy, and canine heart failure (Pun et al., 2023). Because the change is not uniform, the therapeutic direction for S368 remains unresolved.
The role of Cx43 in cancer has also changed with new evidence. It was first regarded as a tumor suppressor because gap junction activity supports apoptosis and limits migration, but recent studies increasingly describe a pro-tumorigenic role in advanced cancers. Cx43 is upregulated in invasive breast carcinomas and lymph node metastases, and high expression is linked to poor prognosis in estrogen receptor-negative breast cancer. In contrast, promoter methylation reduces its expression in non-small cell lung cancer. Many channel-independent effects come from the C-terminal tail, which binds tubulin, ZO-1 and Hsc70 and can increase N-cadherin expression and cell migration without gap junction activity (Paunikar and Tamagnone, 2024).
10. Therapeutic Targeting of Connexin 43
Several Cx43 mimetic peptides have been developed and are based on the extracellular loops (Gap26, Gap27, and Peptide5), the cytoplasmic loop (Gap19 and L2), or the C-terminal tail (JM2, Cx43tat, CycliCX, and the αCT family) (Marsh et al., 2021). The αCT1 peptide was designed to block the interaction between Cx43 and ZO-1 (Pun et al., 2023). αCT1 is the most advanced candidate. In its Granexin formulation, it has been tested in one Phase I and three Phase II trials in chronic wounds, with weekly topical dosing for three months, no severe adverse events and no antibodies detected against the peptide. Phase II testing has covered venous leg ulcers, diabetic foot ulcers and surgical scar mitigation. In preclinical heart models, a single low-dose intravenous dose of Gap26 or Gap27 reduced infarct size in rats by up to 61%, and Gap19 showed anti-arrhythmic effects in mouse models of arrhythmogenic right ventricular cardiomyopathy and Duchenne muscular dystrophy (Marsh et al., 2021).
Cancer offers a second therapeutic setting. In preclinical work, Cx43 knockdown or the gap junction inhibitors tonabersat and meclofenamate reduced brain metastases, and antibodies against the second extracellular loop slowed tumor growth in rat C6 glioma models (Paunikar and Tamagnone, 2024). The same protein can therefore be a target for blocking, as in tumors and injured heart, or for preserving coupling, as in wound repair.
11. Conclusions
Cx43 is a four-pass membrane protein that forms gap junction channels, free hemichannels and, through internal translation, a family of truncated isoforms led by GJA1-20k. Recent cryo-EM structures show a dynamic N-terminal gate whose conformation is influenced by lipids, pH, and the C-terminal tail. The protein turns over within hours, and phosphorylation of its C-terminal tail, especially at S368, controls its permeability, assembly, and degradation. Its presence in mitochondria links it to respiration, reactive oxygen species and cardioprotection. In the ovarian follicle, Cx43 couples granulosa and cumulus cells into a network through which signals reach the oocyte, while Cx37 carries the final step across the oocyte membrane. FSH builds this network, and LH dismantles it through MAPK-dependent phosphorylation, which lowers oocyte cGMP and allows meiosis to resume. In porcine IVM, Cx43 is found on the oolemma, on TZPs and on the zona pellucida, and its level changes during maturation. Cx43 is therefore both a structural part of the cumulus–oocyte partnership and a protein whose dysfunction contributes to skin, heart, and malignant disease.
References
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Connexin 43 (Cx43) is encoded by the gene GJA1, is the most commonly expressed connexin, and is present in most cell types. Cx43 participates in numerous cellular processes, including cardiac contraction, electrical coupling, cell differentiation, and cell death (Lee et al., 2023). The six monomers of a single type of connexin assemble to create a single hemi-channel or "connexon." When two connexons from adjacent cells dock, they create an intercellular channel that then aggregates into a gap junction plaque (Fournier et al., 2025). Ions, metabolites, and second messengers move directly from one cytoplasm to another through these channels without first entering the extracellular space. Cx43 differs from many proteins because it is not an enzyme, hormone, growth factor, or soluble recognition molecule. Instead, it is a four-transmembrane spanning protein whose primary product is a channel. Beyond forming intercellular connections, free hemi-channels can be activated under pathological conditions, and the protein also exists within mitochondria; a smaller fragment produced via alternative splicing of the Cx43 mRNA plays important roles in both trafficking and mitochondrial regulation (Boengler et al., 2022).
Because of its role in reproduction, animal scientists are interested in Cx43. Granulosa cells are primarily connected via Cx43, while connexin 37 (Cx37) is the major connexin forming connections between the oocyte surface and surrounding cells (Turathum et al., 2021). In addition, studies have localized Cx43 to both the oolemma and the transzonal projections of porcine oocytes during in vitro maturation (Lee et al., 2016). This review examines Cx43 from a protein-centered perspective, covering its structure, life cycle, channel modes, isoforms, mitochondrial functions, roles in the cumulus–oocyte complex and porcine IVM, disease associations, and therapeutic targeting.
2. Structural Organization of Connexin 43
Human Cx43 contains 382 amino acids (Lee et al., 2023). Each human Cx43 monomer has four transmembrane helices (TM1-TM4), two extracellular loops (ECL1-ECL2), a single cytoplasmic loop, and both cytoplasmic N- and C-termini. Docking between adjacent cell membranes' hemichannels is mediated by the extracellular loops. Residues 232-382 make up most of the sequence not conserved across connexin proteins and serve as the primary site for post-translational modifications (Pun et al., 2023). Cryo-electron microscopy (Cryo-EM) in 2023 enabled the first high-resolution reconstruction of the human Cx43 gap junction channel. A dodecamer formed by two hexamers of the hemichannel. The N-terminal domain lies parallel to the membrane surface; TM2 is positioned away from the pore center, leaving six peripheral openings and one large central opening. Sterol-like densities appear in the pore and between each pair of N-terminal domains. Molecular dynamics simulations show that these molecules seal the channel, suggesting this structure represents a possible closed state. The cytoplasmic loop and the C-terminal tail were not resolved (Qi et al., 2023).
In addition to providing a dodecameric representation of the channel structure, a second cryo-EM study examined how environmental and mutational changes affected its conformation. This work provided evidence for three forms of the N-terminal alpha-helix (termed gate-covering, pore-lining and flexible intermediate) based on structures determined using cryo-EM. These studies also showed that cholesteryl hemisuccinate shifted the equilibrium to favor an increase in the gate-covering form. In contrast, mutations to the C-terminal region or altered pH shift the equilibrium to favor the pore-lining form. For some of these states, an alpha-to-pi transition in TM1 opens up a secondary pathway into the membrane space (Lee et al., 2023). Figure 1 shows the dodecameric channel and the position of a single monomer within it. Panels A and B show the cryo-EM density map and model of the dodecameric channel at 2.26 Å, with each monomer colored; panel C shows one monomer within the channel. Panel D (alignment with Cx26, Cx46 and Cx31.3).
Figure 1. Cryo-EM structure of the human Cx43 gap junction channel and the position of a single Cx43 monomer.
3. Life Cycle, Turnover and Phosphorylation
Cx43 is synthesized in the endoplasmic reticulum, oligomerized into hemichannels in the Golgi apparatus and delivered to the plasma membrane as closed hemichannels (Boengler et al., 2022). Delivery to cell borders depends in part on GJA1-20k, a short internally translated isoform that acts as a trafficking partner for the full-length protein (Fournier et al., 2025). Once in the membrane, undocked hemichannels collect in the perinexus, the zone surrounding the gap junction plaque. The plaque is renewed continuously. Each gap junction has a half-life of only a few hours, after which internalized connexons are degraded through proteasomal or lysosomal pathways. The C-terminal tail controls much of this cycle. It is phosphorylated, acetylated, S-nitrosylated, ubiquitinated and SUMOylated, and its serine and tyrosine residues are targeted by Src, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC). PKC isozymes phosphorylate serine 368 (S368), which lowers channel permeability and promotes internalization and degradation. PKCγ-driven disassembly also requires zonula occludens-1 (ZO-1), which binds the C-terminal tail near S368 (Pun et al., 2023).
4. Gap Junction Channels and Hemichannels
Cx43 operates in two channel types. Open hemichannels function as part of gap junctions, while closed free hemichannels rest in the plasma membrane. During metabolic blockade or cardiac ischemia, free hemichannels can be opened. Opening of free hemichannels in cardiomyocytes during diastole allows sodium and calcium ions to enter the cell via these channels, each with a single-channel conductivity of approximately 220 pS. This increases membrane depolarization, disturbing normal calcium ion signaling within the cell (Boengler et al., 2022).
Hemichannel clusters near the perinexus area in the heart may serve structural roles. In this zone, Cx43 hemichannel clusters lie near Nav1.5 sodium channels. Ephaptic coupling has been suggested between such adjacent membranes, where tight apposition is required for electrical conduction. Thus, Cx43 participates in tissue function through both docked channels and free hemichannel openings, as well as the membrane zones surrounding these openings.
5. Internally Translated Isoforms and GJA1-20k
The GJA1 messenger RNA produces more than one protein. Internal translation from downstream methionines generates six N-terminally truncated isoforms, named by molecular weight as GJA1-32k, GJA1-29k, GJA1-26k, GJA1-20k, GJA1-11k and GJA1-7k. GJA1-20k starts at methionine 213 and is usually the most abundant. It contains part of TM4 and the entire C-terminal tail, so it cannot form a hemichannel, but it retains the tail's regulatory features. Cardiac studies first linked GJA1-20k to delivery of full-length Cx43 to the plasma membrane, cytoskeletal dynamics, and mitochondrial fission and distribution. GJA1-20k is also expressed in cancer cells, where it may contribute to epithelial-to-mesenchymal transition and metastatic behavior (Fournier et al., 2025).
6. Mitochondrial Connexin 43
Cx43 is also located within mitochondria, primarily found in the subsarcolemmal mitochondria of ventricular cardiomyocytes. Import into the mitochondria occurs via a pathway that uses the chaperone Hsp90 and the outer-membrane translocase (TOM), and most data indicate it resides at the inner mitochondrial membrane. GJA1-20k is localized at the outer membrane of the mitochondria. Mitochondrial Cx43 appears to allow potassium ions to enter, affecting respiration and reactive oxygen species production. Both ischemic and diazoxide preconditioning use mitochondrial Cx43 to some extent; however, its role in post-conditioning is poorly understood. The estimated conductance of mitochondrial Cx43 hemichannels is about 130 pS, lower than that of plasma membrane hemichannels (Boengler et al., 2022). GJA1-20k provides an additional level of mitochondrial organization, as it has been linked to mitochondrial fission and subcellular localization (Fournier et al., 2025). Thus, these two forms affect mitochondria differently. Figure 2 summarizes the import route, membrane location, and proposed functions of mitochondrial connexins. It shows the Hsp90/TOM import pathway, Cx43's effects on oxidative phosphorylation, potassium entry, and reactive oxygen species, and the outer-membrane location of Gja1-20k.
Figure 2. Localization, import and proposed functions of mitochondrial Cx43, including the outer-membrane position of GJA1-20k.
7. Connexin 43 in the Cumulus–Oocyte Complex
Cumulus cells reach the oocyte through transzonal projections (TZPs), thin cytoplasmic extensions that cross the zona pellucida and form gap junctions at their tips. Three-dimensional electron microscopy of mouse follicles showed that most TZPs do not reach the oocyte. Each cumulus cell carried, on average, 31 ± 5 free-ended TZPs and 9 ± 2 connected TZPs, and 78% of TZP contacts were between projections from different cells, some of which were confirmed as gap junctions (Baena and Terasaki, 2019). The TZP network is therefore also a network of cumulus-to-cumulus contacts. The two main follicular connexins are divided as follows: Cx43 is found mainly in granulosa and cumulus cells. It is thought to mediate communication between cumulus cells, whereas Cx37 is found in the oocyte and cumulus cells and is thought to mediate cumulus-to-oocyte communication. In mice lacking Cx37, Cx43 expressed in the oocyte restored cumulus–oocyte complex formation and fertility, although Cx37 channels differ in their selectivity for larger molecules and ions (Xie et al., 2023).
Gonadotropins regulate Cx43 in opposite directions. Follicle-stimulating hormone (FSH) increases Cx43 expression and gap junction formation. Luteinizing hormone (LH) activates MAPK-dependent phosphorylation of Cx43, which closes the junctions and reduces the protein; in bovine cumulus cells, Cx43 messenger RNA falls about six hours after the LH surge. Gap junctions control the movement of cyclic GMP (cGMP) and cyclic AMP (cAMP) into the oocyte, and the fall in oocyte cGMP after junction closure allows cAMP hydrolysis and the resumption of meiosis (Kordowitzki et al., 2021). Upstream of these events, LH acts on mural granulosa cells to release epidermal growth factor (EGF) receptor ligands, which activate ERK/MAPK signaling in cumulus cells and drive actin reorganization and TZP retraction (Turathum et al., 2021). Figure 3 summarizes how FSH and LH regulate Cx43 and Cx37 channels before ovulation and during meiotic resumption. Panel A shows FSH-driven channel synthesis and LH-driven disruption in the antral follicle; panel B shows LH-induced Cx43 phosphorylation, Cx37 closure and the cAMP and maturation-promoting factor changes that lead to germinal vesicle breakdown.
Figure 3. Regulation of Cx43- and Cx37-based gap junction communication in the antral follicle before ovulation and during meiotic resumption.
8. Connexin 43 in Porcine Oocyte Maturation
Porcine cumulus-oocyte complexes have been widely used to study Cx43. Most research to date has focused on these complexes as an integrated system rather than on individual components. Maturing porcine complexes in NCSU23 medium and analyzing them at 0, 14, 28, and 42 hours revealed that the Cx43 signal was present on the oolemma, TZPs, and the surface of the zona pellucida. Following maturation, Cx43 band density initially increased until 14 hours post-maturation, after which it began to decline (Lee et al., 2016). The observed changes in Cx43 band density correlate well with expected changes in functional connectivity as the oocyte resumes meiotic progression. In addition to disrupting communication between cumulus cells and the oocyte, FSH stimulates the release of EGF-like factors, including amphiregulin (AREG) and epiregulin (EREG), which signal from granulosa cells through cumulus cells to the oocyte (Turathum et al., 2021). Therefore, in porcine IVM systems, gonadotropin supplementation directly influences the pathway(s) that regulate Cx43. Culturing porcine cumulus-oocyte complexes under conditions of stress can alter cumulus cell-oocyte interactions. Heat exposure in porcine IVM cultures significantly reduced the percentage of oocytes reaching MII by 44 hours (from 77% to 6%), along with reductions in TZP numbers and TZP F-actin content. mRNA levels of β-actin and γ-actin also decreased in cumulus cells, and colocalization between TZPs and gap junctions was lower than when identified using antibodies against connexin 45 (Yin et al., 2020). To date, few studies have measured the direct effects of mycotoxin exposure (e.g., deoxynivalenol) on Cx43 localization or TZP density in porcine complexes; however, cumulus expansion and nuclear maturation depend on effective signaling between cumulus cells.
9. Connexin 43 in Disease
Mutations in GJA1 or misregulation of Cx43 cause or contribute to oculodentodigital dysplasia, palmoplantar keratoderma, heart disease and cancer (Lee et al., 2023). In the heart, the direction of change depends on the condition. S368 phosphorylation increases in some models of ischemia–reperfusion, myocarditis, and arrhythmia, but decreases in furazolidone-induced dilated cardiomyopathy, plakophilin-2-knockout arrhythmogenic right ventricular cardiomyopathy, and canine heart failure (Pun et al., 2023). Because the change is not uniform, the therapeutic direction for S368 remains unresolved.
The role of Cx43 in cancer has also changed with new evidence. It was first regarded as a tumor suppressor because gap junction activity supports apoptosis and limits migration, but recent studies increasingly describe a pro-tumorigenic role in advanced cancers. Cx43 is upregulated in invasive breast carcinomas and lymph node metastases, and high expression is linked to poor prognosis in estrogen receptor-negative breast cancer. In contrast, promoter methylation reduces its expression in non-small cell lung cancer. Many channel-independent effects come from the C-terminal tail, which binds tubulin, ZO-1 and Hsc70 and can increase N-cadherin expression and cell migration without gap junction activity (Paunikar and Tamagnone, 2024).
10. Therapeutic Targeting of Connexin 43
Several Cx43 mimetic peptides have been developed and are based on the extracellular loops (Gap26, Gap27, and Peptide5), the cytoplasmic loop (Gap19 and L2), or the C-terminal tail (JM2, Cx43tat, CycliCX, and the αCT family) (Marsh et al., 2021). The αCT1 peptide was designed to block the interaction between Cx43 and ZO-1 (Pun et al., 2023). αCT1 is the most advanced candidate. In its Granexin formulation, it has been tested in one Phase I and three Phase II trials in chronic wounds, with weekly topical dosing for three months, no severe adverse events and no antibodies detected against the peptide. Phase II testing has covered venous leg ulcers, diabetic foot ulcers and surgical scar mitigation. In preclinical heart models, a single low-dose intravenous dose of Gap26 or Gap27 reduced infarct size in rats by up to 61%, and Gap19 showed anti-arrhythmic effects in mouse models of arrhythmogenic right ventricular cardiomyopathy and Duchenne muscular dystrophy (Marsh et al., 2021).
Cancer offers a second therapeutic setting. In preclinical work, Cx43 knockdown or the gap junction inhibitors tonabersat and meclofenamate reduced brain metastases, and antibodies against the second extracellular loop slowed tumor growth in rat C6 glioma models (Paunikar and Tamagnone, 2024). The same protein can therefore be a target for blocking, as in tumors and injured heart, or for preserving coupling, as in wound repair.
11. Conclusions
Cx43 is a four-pass membrane protein that forms gap junction channels, free hemichannels and, through internal translation, a family of truncated isoforms led by GJA1-20k. Recent cryo-EM structures show a dynamic N-terminal gate whose conformation is influenced by lipids, pH, and the C-terminal tail. The protein turns over within hours, and phosphorylation of its C-terminal tail, especially at S368, controls its permeability, assembly, and degradation. Its presence in mitochondria links it to respiration, reactive oxygen species and cardioprotection. In the ovarian follicle, Cx43 couples granulosa and cumulus cells into a network through which signals reach the oocyte, while Cx37 carries the final step across the oocyte membrane. FSH builds this network, and LH dismantles it through MAPK-dependent phosphorylation, which lowers oocyte cGMP and allows meiosis to resume. In porcine IVM, Cx43 is found on the oolemma, on TZPs and on the zona pellucida, and its level changes during maturation. Cx43 is therefore both a structural part of the cumulus–oocyte partnership and a protein whose dysfunction contributes to skin, heart, and malignant disease.
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