The skin serves as a barrier, protecting the human body from external threats. Primary functions of skin tissue include preventing water loss, regulating temperature, forming a barrier to facilitate molecular exchange and maintaining immune surveillance (García-Vega et al., 2019; Anderton and Alqudah, 2022). There are three layers in the skin; the epidermis, the dermis and the hypodermis with the epidermis and dermis separated by the dermoepidermal junction, including a basal lamina (Czyz et al., 2023). The epidermis, the outermost layer of the skin, is an avascular tissue and is composed primarily of keratinocytes (Faniku et al., 2015; García-Vega et al., 2019). This layer is further subdivided into four distinct layers, each with specific features, starting from the innermost layer to the outermost, i.e., the stratum basale, the stratum spinosum, the stratum granulosum, and the stratum corneum. The basal layer contains proliferating keratinocytes that are able to divide to produce committed cells migrating towards the upper layers of the epidermis and undergoing a differentiation process leading to a specialized terminally state named corneocyte (Fuchs, 2008; Martin et al., 2014; Faniku et al., 2015). During epidermal differentiation, keratinocytes undergo significant modifications in both protein expression and structural organization, which are orchestrated by complex signaling pathways, basement membrane components, calcium balance, and adhesion molecules (Fuchs, 2008). In addition, a calcium ion (Ca²⁺) gradient exists within the epidermis, increasing from the basal layer to the granular layer (Tu et al., 2012; Adams et al., 2015). This Ca²⁺ gradient plays a crucial role in a plethora of cellular pathways during keratinocyte differentiation process. Moreover, both intracellular and extracellular Ca²⁺ regulates signaling pathways as well as the expression and function of adhesion molecules such as integrins that connect keratinocytes to each other and to the basement membrane (Pani and Singh, 2008). The Ca²⁺ gradient is critical for keratinocyte differentiation and skin barrier formation through activity of transcription factors such as activator protein 1 (AP-1) that induces keratinocyte-specific differentiation genes such as involucrin and profilaggrin (Ng et al., 2000). Moreover, Ca²⁺ signaling has been shown to preserve the integrity of both the physical and anti-bacterial barrier of the stratum corneum by regulating lamellar body secretion and cytokine expression, respectively (Choi and Yoon, 2014; Lee and Lee, 2018; Srinivas et al., 2018).

Because the epidermis lacks blood vessels, the coordination and the communication among epidermal cells are ensured by intercellular junctions. One crucial mechanism for this communication is gap junctions (GJs), which are specialized connections enabling the direct intercellular communication between cytoplasm of adjacent cells. In this article, we will explore into the formation and regulation of the GJ channels, specifically examining the functions and the consequences of gene mutations in connexin 26 (Cx26), Cx30, Cx30.3, Cx31, and Cx43, which have been linked to various skin disorders. Moreover, we will provide a concise overview of the dysregulation of connexins in other dermatological conditions, offering valuable insights into the role of connexins in maintaining healthy skin and the essential role of gap junctional intercellular communication (GJIC) in this process.

GJs facilitate the transport of ions (Ca²⁺ and K⁺), small metabolites (glucose, vitamins, and amino acids), secondary messengers (cAMP and ATP), and short interfering RNAs between neighboring cells (Figure 1) (Goodenough and Paul, 2009; Aypek et al., 2016; Garcia-Vega et al., 2021). GJs are formed by connexins (Figure 1A) that assemble into hemichannels or connexons (Figure 1B), which then can dock with connexons from neighboring cells to mediate GJIC on the plasma membrane (Figure 1C). This intercellular communication plays a pivotal role in numerous physiological processes, including cell proliferation, differentiation, and homeostasis (Anderton and Alqudah, 2022). Consequently, any disruption in connexin function or GJIC can lead to diseases.

Connexins are the main structural subunits of GJ channels in chordates, and possess an intracellular loop (IL), an amino (N) and a carboxyl (C) domains, along with four conserved transmembrane helical domains and two extracellular loops (EL1 and EL2) (Figure 1A) (Martin and van Steensel, 2015). Connexons are formed in the endoplasmic reticulum (ER)—Golgi network through the oligomerization of six connexins, which can assemble either into homomeric hemichannels, composed of the same type of connexins, or heteromeric connexons, assembled by different types of connexins (Figure 2i). Following trafficking to the plasma membrane, hemichannels can dock head-to-head with other hemichannels from neighboring cells to complete the formation of the GJs, hence facilitating the intercellular communication (Figure 2ii-iv). Hemichannels can also function independently in non-junctional areas, mediating the exchange of molecules across the plasma membrane (Figure 2iii). Additionally, connexins can influence gene expression, cellular migration, and other molecular mechanisms by localizing to the cytoplasm and the nucleus without forming channels on the plasma membrane (Kotini et al., 2018; Martins-Marques et al., 2023). Connexins have a short half-life and the turnover of the hemichannels and GJs, which is mediated by endocytosis at the plasma membrane, enables cells to rapidly respond to environmental changes (Figure 2) (VanSlyke and Musil, 2005; Falk et al., 2016; Aasen et al., 2019). After GJ endocytosis, connexins follow various intracellular routes before lysosomal degradation (Figures 2v–2viii). These routes include fusion of connexosomes with early endosomes (Figure 2vi), enclosure by autophagosomes followed by fusion with lysosomes, and direct fusion with lysosomes (Figure 2viii) (Carette et al., 2015; Totland et al., 2020).

GJs regulate cellular homeostasis by facilitating the transport of molecules with molecular weights below 1.4 kDa (Sanchez and Verselis, 2014; Leybaert et al., 2017; Bai et al., 2018). Due to their critical functions, connexin expression and channel activity are tightly regulated by various factors, including extracellular Ca²⁺, intracellular pH, membrane potential, post-translational modifications, and epigenetic regulation (Aasen et al., 2018; 2019). Moreover, non-coding RNAs including microRNAs can also regulate connexin expression (Curcio et al., 2013; Zeng et al., 2021). Extracellular Ca²⁺ exerts a significant influence on connexin channel gating by directly binding to the channels. For example, high Ca²⁺ concentrations constrict the diameter of the Cx26 channel pore lumen (Lopez et al., 2016), maintaining these hemichannels in a predominantly closed state (Chen et al., 2022). Cx26 and Cx30 hemichannels possess specific residues, such as D50 and K61, which form salt bridges at the extracellular entrance of the pore. Ca²⁺ binding to these residues disrupts the salt bridge, promoting the closed state of Cx26 and Cx30 hemichannels (Lopez et al., 2016). Connexins, especially Cx26, display a distinct distribution of charged amino acid residues: the cytoplasmic domain is positively charged, while the transmembrane and extracellular domains are negatively charged (Maeda et al., 2009). Within the pore lining, negatively charged residues form an electrostatic network that is essential for hemichannel gating and Ca²⁺ binding. Beyond these direct effects, Ca²⁺ regulates its own transport by interacting with the Ca²⁺ binding protein, namely, calmodulin. Calmodulin binding was shown to induce Cx43 channel closure, indicating the involvement of Ca²⁺/calmodulin mechanisms in channel gating (Xu et al., 2012).

Connexin channels exhibit sensitivity to changes in environmental pH. A decrease in pH triggers the closure of Cx43 channels, a process mediated by conformational changes in the cytoplasmic domains, which act as gating plugs in response to acidic conditions (Peracchia, 2004). Similarly, channel closure under acidic conditions is observed in Cx26 due to conformational changes (Wang et al., 2012). In this instance, the pore lumen is occluded through voltage-driven alterations in its N-terminal domain (Maeda et al., 2009; Brotherton et al., 2022). Moreover, voltage-dependent conformational changes serve as a crucial regulator of connexin channel gating which is induced by a potential difference either between the extracellular and intracellular spaces or between the cytoplasm of neighboring cells (Bargiello et al., 2012; Oh and Bargiello, 2015). For example, Cx26 hemichannels become active at −50 mV, with their activation increasing upon depolarization until they become inactive at positive membrane potentials (González et al., 2006). Voltage-driven alterations in the shape of ion channels, achieved by modifying the positions of charged amino acids, are linked to other structural alterations including destabilization of channel pore parahelix segment that determine whether the channel pore is open or closed (Bargiello et al., 2018).

Connexins harbor multiple sites susceptible to post-translational modifications, such as phosphorylation, S-nitrosylation, and SUMOylation, which can modulate channel functions in diverse ways. Phosphorylation of serine, threonine, or tyrosine residues within connexins can influence their assembly and trafficking, whereas S-nitrosylation induces structural changes in connexin proteins by altering the cysteine oxidation state (Saez et al., 2003). Small ubiquitin-like modifier (SUMO) proteins can conjugate with lysine residues of connexins and regulate GJ conformation and trafficking (Kjenseth et al., 2012; Aasen et al., 2018). Furthermore, connexin expression can be regulated through epigenetic mechanisms and dysregulation of epigenetic processes is indicated in various diseases (Vinken, 2016; Aasen et al., 2018). For example, Cx43 downregulation in breast cancer was attributed to the hypermethylation of its promoter region (Yi et al., 2007). In addition, inhibition of histone deacetylase caused the elevation of Cx43 expression and facilitated its translocation to the plasma membrane in tumorigenic cells (Oyamada et al., 2005), highlighting the involvement of epigenetic regulatory mechanisms for control of connexin expression (Aasen et al., 2018). As briefly summarized, connexin channel function represents a complex process tightly regulated by a multitude of factors. These regulatory mechanisms ensure appropriate connexin function in response to diverse cellular and environmental cues.

Connexin expression patterns in the epidermis vary considerably across its distinct layers, playing a crucial role in maintaining the epidermal homeostasis and renewing the skin. The basal layer, harboring the epidermal basal cell pool responsible for replenishing the upper cell layers, maintains a delicate balance between the rate of cell differentiation in this layer and the rate of terminally differentiated cells reaching the epidermis’ outer surface (Fuchs, 2008). Throughout the epidermal differentiation process, immunohistochemical analysis revealed that at least ten distinct connexins are expressed within the various layers of the epidermis. In the basal layer, cells primarily express Cx43, while Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx40, Cx43, and Cx45 are observed in the spinous layer. Transitioning to the granular layer, Cx30.3, Cx31, and Cx43 are expressed at high levels, whereas Cx26, Cx31.1, Cx40, and Cx45 are found at lower levels (Figure 3) (Martin et al., 2014; Zhang and Cui, 2017). Beyond their specific distribution in the epidermis, there is a link between various skin disorders and mutations in Cx26, Cx30, Cx30.3, Cx31, and Cx43, suggesting their substantial roles in establishing and maintaining the integrity of the epidermal barrier (García-Vega et al., 2019; Lucaciu et al., 2023).

The functional significance of different connexin isoforms expressed in the epidermis is still being investigated. For example, Cx43, which is predominantly expressed in most layers, particularly the basal layer, regulates both proliferation and differentiation of keratinocytes. Cx43 interacts with junctional proteins, including the tight junction molecules, zona occludens, and adherens junction components beta-catenin, to regulate proliferation and differentiation (Scott et al., 2012). In addition, Cx26 and Cx30 channels play a regulatory role in processes such as wound healing within the granular and spinosum layers of the epidermis by influencing protein pathways associated with proliferation through the passage of molecules such as ATP and Ca²⁺ (Essenfelder et al., 2004; Srinivas et al., 2018). The precise function of each connexin isoform within the epidermis remains elusive. Therefore, unveiling the effects of skin-associated connexin gene mutations can provide valuable insights into their function in maintaining epidermal homeostasis.

The epidermis lacks blood vessels, so it relies on GJIC between keratinocytes for the coordination of signals and molecule supply to maintain epidermal homeostasis (Chanson et al., 2018; Qiu et al., 2022). Humans express at least ten distinct connexin isoforms (Mese et al., 2011; Garcia-Vega et al., 2021), and at least five of these have been implicated in eleven human skin diseases (Table 1). Six of these diseases are caused by mutations in the GJB2 gene, which encodes for the Cx26 protein (Zhang and Cui, 2017; Sellitto et al., 2021). In addition to Cx26, mutations in GJB6, GJB4, GJB3 and GJA1, encoding Cx30, Cx30.3, Cx31 and Cx43, respectively have also been linked to diverse epidermal diseases (Avshalumova et al., 2014; Laird and Lampe, 2022). Disease-associated mutations in connexin genes can disrupt GJIC in various ways, leading to diverse phenotypes. These mutations can lead to the formation of aberrant hemichannels, allowing uncontrolled passage of molecules across the plasma membrane. Mutations can also result in loss of function, where the connexin protein is unable to form functional GJs. Additionally, mutations can cause mislocalization of connexin proteins within the cell, hence preventing them from reaching the cell membrane and forming GJs. Finally, they can have a dominant-negative effect on the GJIC facilitated by other connexins (Figure 4) (Scott et al., 2012). Here, we will first discuss the Cx26-associated epidermal diseases followed by Cx30.3, Cx31, Cx43 and Cx30 skin diseases.

While mutations in connexin genes can directly cause skin diseases, their expression is also dysregulated in other skin conditions, including psoriasis, pressure ulcers, wound healing issues, and even skin cancers (Garcia-Vega et al., 2021). Psoriasis, an inflammatory skin disorder characterized by thickened and scaly lesions, serves as a major example. Studies revealed a dramatic upregulation of Cx26 in psoriatic lesions, suggesting it as a marker for psoriasis (Rivas et al., 1997; Labarthe et al., 1998; Lucke et al., 1999; Sun et al., 2010; Li et al., 2014; Martin et al., 2014; Yao et al., 2015; Garcia-Vega et al., 2021). Increased Cx26 expression by stimulation of pro-inflammatory responses promoted the release of inflammatory molecules such as ATP and IL-6 into the extracellular environment, indicating the contribution of elevated hemichannels in this process (Garcia-Vega et al., 2019). Additionally, mice engineered to overexpress Cx26 in keratinocytes exhibited psoriasis-like phenotypes, supporting the link between Cx26 and impaired barrier function (Djalilian et al., 2006).

Cx43 presents a more complex picture in psoriasis. Its levels were slightly elevated in some studies (Labarthe et al., 1998), while others reported a decrease in psoriatic lesions (Liang et al., 2019). This discrepancy might be explained by recent findings suggesting that Cx43 in psoriatic tissues underwent post-translational modifications, especially phosphorylation that might interfere with Cx43 biogenesis rather than increased mRNA production (O’Shaughnessy et al., 2021). Interestingly, IL-22, a cytokine involved in psoriatic keratinocyte hyperproliferation, seems to downregulate Cx43 expression, further hindering GJIC (Liang et al., 2019).

Connexins have garnered significant research interest due to their involvement in various diseases. Over the years, therapeutic strategies targeting connexin function have emerged, including non-peptide chemicals, peptide mimetics, antibodies, allele-specific siRNAs (AS-siRNAs), and antisense oligodeoxynucleotides (asODNs) (Table 2). Connexin channel inhibitors, in particular, have played a key role in elucidating their function through channel activity modulation. Carbenoxolone (CBX), a non-selective GJ blocker, has been a pillar since 1986 (Davidson et al., 1986). Its ability to reduce fluorescent dye uptake in KID syndrome-associated Cx26 mutant cells (Aypek et al., 2016; Taki et al., 2018) highlighted its potential in investigating hyperactive channel function. However, its non-specificity and side effects limit its therapeutic application (Tovar et al., 2009; Connors, 2012; Buckley et al., 2021). Mefloquine, an antimalarial drug, effectively blocks Cx50 and Cx36 GJ channels (Cruikshank et al., 2004) and showed promise in inhibiting KID syndrome-associated Cx26 mutations (Levit et al., 2015). Its lipophilicity facilitates topical delivery, but its approved indications and potential neuropsychiatric concerns require cautious consideration (Weinke et al., 1991). Boldine, an alkaloid, specifically inhibits Cx43 GJs and holds potential for diabetic wound healing (Hernández-Salinas et al., 2013). Similarly, tonabersat, a small molecule targeting Cx43 hemichannels (Lyon et al., 2020), demonstrated efficacy in reducing inflammation and maintaining vascular integrity in retinal disease models (Mat Nor et al., 2020). These studies showcase the diverse array of connexin inhibitors utilized to investigate connexin channel function. However, further research is warranted to address their specificity and potential side effects in complex biological systems for clinical applicability.

Driven by the non-specific nature of connexin and channel inhibitors, researchers have actively explored novel strategies for selectively targeting specific connexin proteins and their associated channels. The six cysteine residues within the extracellular domains of connexins play a critical role in GJ assembly by forming disulfide bonds with other cysteines. This disulfide exchange is thought to be essential for channel docking and opening. Understanding the function of these cysteines has facilitated the design of peptides that can mimic connexin interactions during GJ channel formation. Additionally, variable amino acids within the extracellular loops influence the specificity of these interactions, enabling the development of connexin-specific peptides (Dahl et al., 1992). Short peptides containing conserved motifs in the extracellular loops involved in junction formation were synthesized (Warner et al., 1995) and peptides containing the SRPTEK motif in the EL2 and amino acids in the putative membrane-spanning region effectively altered GJ formation. Moreover, mimetic peptides containing this motif effectively delayed GJ formation.

Connexin mimetic peptides, Gap26 and Gap27 containing the SHVR and SRPTEK motifs, respectively, were used to investigate the functionality and expression of connexins in vascular cells (Warner et al., 1995). Importantly, these peptides did not impair de novo connexin expression or trafficking. Another study examined the potential of these peptides for wound healing by studying their effects on human epidermal keratinocytes and dermal fibroblasts in vitro. Blocking GJIC using these peptides increased cell migration rates, suggesting a promising avenue for promotion of wound healing (Wright et al., 2009). Further research revealed that Gap26 and Gap27 achieved their effects by preventing hemichannel docking and promoting cell movement (Lorraine et al., 2015). Specifically, Gap27 was tested as a potential wound healing agent in three settings: primary human corneal epithelial cells (in vitro), whole human corneas (ex vivo), and a rat wound healing model (in vivo). Applying Gap27 accelerated wound closure and corneal cell layering without affecting the release of the inflammatory cytokines IL-6 and TNF-α. After 7 days, Gap27 increased early granulocyte infiltration and the expression of TNF-α and TGF-β, but it did not prevent neovascularization (Elbadawy et al., 2016).

Peptide5, another extensively studied connexin mimetic peptide, targets a region within the EL2 of Cx43 and harbors the SRPTEK motif. At low concentrations, Peptide5 effectively blocked hemichannel opening without disrupting GJIC. However, at higher concentrations, it disrupted the function of pre-existing junctions (O'Carroll et al., 2008). Intriguingly, Cx43 hemichannels have been implicated in secondary lesion spread, a process where the opening of a dysfunctional pore leads to cell death at lesions and exacerbates secondary lesion formation. Peptide5 has been shown to effectively prevent lesion spread and reduce vascular permeability. Furthermore, Peptide5 exhibited anti-inflammatory properties and enhanced neuron survival in various models of brain, retina ischemia-reperfusion injury, and spinal cord injuries (Kim et al., 2017).

αCT1, a Cx43 C-terminal mimetic peptide, effectively inhibits Cx43 hemichannel activity. Harboring the Cx43 zonula occludens-1 (ZO-1) binding domain, αCT1 disrupted their interactions, leading to the sequestration of Cx43 hemichannels from the perinexus regions (Rhett et al., 2011). This consequently reduced the hemichannel density on the plasma membrane. In addition, αCT1 also affected the interaction of Cx43 with other molecules, which in turn altered its phosphorylation status (O'Quinn et al., 2011). αCT1 was shown to hold promise for wound healing. Its application improved the wound closure, promoted the epidermal complexity, decreased the area of granulation tissue, and enhanced the strength and extensibility of the skin in mouse models by attenuation of neutrophil infiltration and changing the epidermal Cx43 organization (Ghatnekar, et al., 2009). Additionally, αCT1 increased the rate of wound closure and reduced inflammation in excisional wounds that were recovered with decreased discoloration (Ongstad et al., 2013). The efficacy and safety of αCT1 was assessed in chronic diabetic wound healing with randomized and multi-center clinical trials. It was observed that the ulcer area was reduced by 94% within 12 weeks of treatment without immunogenic or negative consequences (Grek et al., 2015). In venous leg ulcers, the upregulation of Cx43 expression was observed in the edges of wounds and the incorporation of αCT1 in standard of care treatment greatly reduced the ulcer area in 12 weeks when compared to compression bandage therapy alone (Ghanetkar et al., 2015). The benefit of αCT1 was also evident in human incisional wounds as incisions treated with αCT1 mimetic peptide showed 47% improvement in scar scores at 9 months with progression in scar pigmentation, and roughness of the surface without adverse effects (Grek et al., 2017). The basis for the effectiveness of the αCT1 on the wound was shown to be through the alteration of the organization of collagen bundles in dermal granulation tissue, resulting in its random distribution similar to unwound tissue. This randomness was suggested to interfere with the migration of cells in the wound, accelerating the healing rate (Montgomery et al., 2021). Among Cx43 mimetic peptides only αCT1 (Granexin) is undergoing phase 3 clinical trials for skin wounds without local or systemic adverse effects (Montgomery et al., 2018).

Gap19, derived from the cytoplasmic loop of Cx43, selectively blocks Cx43 hemichannels without hindering GJ closure. This blockade is achieved by preventing interactions between the C-terminus and cytoplasmic loop (Wang et al., 2013). Human gingival wounds that heal faster than skin wounds had distinct hemichannel and GJ plaques where Cx43 hemichannels were implicated in expression of wound healing-associated genes (Tarzemany et al., 2017). Moreover, inhibition of Cx43 hemichannels with Gap19 in human gingival fibroblasts modulated the expression of genes involved in wound healing, leading to faster repair and also suggested targeting connexin hemichannels as an additional component in wound healing process (Tarzemany et al., 2017; Tarzemany et al., 2018).

A monoclonal antibody (mAb), abEC1.1, that targets Cx26 hemichannels was identified in a combinatorial library of human single-chain fragment variable antibodies and characterized using organotypic cultures of mouse cochlea, HeLa, and HaCaT cell lines (Xu et al., 2017). In this study, abEC1.1 effectively inhibited hyperactive Cx26-G45E and Cx26-D50N mutant channel activity. To validate the efficacy of abEC1.1 for the treatment of KID syndrome caused by hyperactive Cx26 mutant channels, Peres et al. employed adeno-associated virus-mediated abEC1.1 monoclonal antibody gene transfer treatment in the mouse model. The treatment effectively blocked Cx26-G45E mutant hemichannel activity in the epidermis, leading to an improvement in skin pathology associated with KID syndrome. Without affecting Cx26 expression in the lesional epidermis, abEC1.1 mAb reduced epidermal thickening and decreased the size and number of keratinocytes. Additionally, abEC1.1 treatment restored the expression of keratins (Peres et al., 2023). Similar to Cx26 KID syndrome model, abEC1.1 also blocked the Cx30-A88V hyperactive hemichannels in Clouston syndrome mouse model (Kuang et al., 2020). The topical or systemic administration of the antibody reduced the proliferation of skin cells and decreased the size of sebaceous glands, suggesting the therapeutic potential of abEC1.1 for treating skin pathologies resulting from hyperactive connexin hemichannels.

AS-RNAi is a highly specific technique that can distinguish between two mRNA sequences differing by just one nucleotide. This strategy holds a promise for silencing mutated alleles that cause dominant inherited diseases. In a landmark study published in 2015, Trochet et al. reported the first clinical trial of AS-RNAi therapy for Pachyonychia congenita, an autosomal dominant condition primarily affecting nails and hand skin (Trochet et al., 2015). The encouraging results have opened up avenues for potential applications in other dominant inherited diseases (Smith et al., 2006; Trochet et al., 2015). Building on this approach, Lee et al. developed AS-siRNAs and applied them to an immortalized KID syndrome cell line (KID-KCs) derived from a patient harboring a heterozygous Cx26-D50N mutation, the most prevalent KID syndrome-associated mutation in the GJB2 gene, leading to aberrant hemichannel function (Lee et al., 2020). Among several candidates, S7 AS-siRNAs exhibited the most potent knockdown efficacy, inducing a 63% reduction in total Cx26 mRNA levels and a 56% decrease in Cx26 protein expression in KID-KCs without affecting the expression of the wild-type allele. Additionally, neurobiotin uptake assays revealed a significant decline in hemichannel activity following AS-siRNA application (Lee et al., 2020). Further investigation is warranted to fully elucidate the potential of AS-siRNAs on patients, as well as on the expression of other genes to evaluate any potential side effects. Overall, this study offers valuable insights into the development of AS-RNAi as a therapeutic strategy for skin pathologies associated with dominant mutations arising from single nucleotide changes.

Finally, inhibition of Cx43 using asODNs demonstrated efficacy in treatment of wounds. Mori et al. showed that asODN application in mice reduced Cx43 mRNA level, leading to increased proliferation and migration of keratinocytes and fibroblasts, decreased inflammation, and enhanced granulation tissue formation (Mori et al., 2006). Similarly, sustained release of Cx43-asODN via collagen scaffolds improved wound healing in rats (Gilmartin et al., 2016). Moreover, targeting Cx43 expression with Nexagon, a Cx43-asODN developed by Ocunexus, accelerated wound healing in Phase I and II clinical trials (details unavailable). Notably, Ormonde et al. showed Nexagon efficiently promoted wound repair and epithelial regeneration in non-healing corneal wounds, with a single application stimulating early limbal vessel recovery and corneal re-epithelization within 18 h, showing the efficacy of asODN in different types of wounds (Ormonde et al., 2012). Similar to Cx43, Cx26-asODN-coated collagen scaffolds inhibited the rise of Cx26 protein at the edge of excisional wounds and promoted healing in rats, suggesting the potential of asODNs for treating skin conditions with elevated connexin levels (Phillips et al., 2018).

The field of connexin-targeting therapeutics has witnessed significant advancements, including the development of hemichannel/GJ inhibitors, mimetic peptides, allele-specific RNA interference, and asODNs. Recently, specific antibodies such as abEC1.1 have shown promising potential in treating skin pathologies associated with connexin mutations. This progress holds great promise for the development of targeted therapies and novel side-effect-free techniques for diseases resulting from connexin mutations or altered connexin expression.

The diverse expression patterns of connexins across all epidermal layers and cell types underpin their essential role in maintaining skin health. Moreover, the association of various skin disorders with diverse phenotypes caused by connexin gene mutations supports the importance of each connexin. However, the unique function of each isoform during epidermal homeostasis is not exactly known (Table 1). Functional characterization of individual connexin mutations has provided valuable insights into their effects on protein function and their roles in the skin. These studies reveal that skin-associated connexin mutations can disrupt protein function in several ways, depending on the type and location of the alteration within the polypeptide sequence. These disruptions can include: defects in the biosynthetic pathway, impaired GJIC, dominant-negative effects on wild-type connexins, and the formation of hyperactive hemichannels (Figure 4). Impaired GJIC, either due to the mutant connexins themselves or their dominant-negative effect on wild-type connexins, can alter the type and extent of communication between cells, potentially affecting proliferation and differentiation processes. Furthermore, the generation of hyperactive hemichannels on the plasma membrane can facilitate uncontrolled molecular exchange between the cytoplasm and the extracellular environment, particularly for signaling molecules such as Ca²⁺ and ATP. Increased intracellular Ca²⁺ levels can modify the activity of several Ca²⁺-dependent signaling processes, interfering with epidermal homeostasis. Hemichannels can also release molecules like ATP into the extracellular environment. This leakage can not only promote cell death but also influence the behavior of surrounding cells by acting as paracrine signals that activate purinergic receptors or other mechanisms, ultimately disrupting cell function. Finally, the retention of mutant proteins in the cytosol can impair cellular structures and alter the expression of molecules such as AP-1 or TGF-β1, which are involved in maintaining keratinocyte homeostasis. Additionally, connexin expression is dysregulated in various skin conditions such as psoriasis, chronic wounds, and cancers. Ultimately, these disruptions interfere with keratinocyte proliferation and differentiation, thereby contributing to the development of various epidermal disorders. While the general impact of connexin mutations or dysregulated expression on epidermal homeostasis has been started to be understood, the exact underlying mechanisms and the effects of connexin alteration on cellular processes that lead to epidermal changes remain largely unknown. Thus, deciphering these molecular and cellular changes arising from abnormal connexin function involved in keratinocyte proliferation and differentiation processes is crucial for better understanding the pathogenesis of each skin condition, potentially leading to the development of more precise therapies for treatment or symptom management.