Pado is a dual-function protein created by fusing an engineered voltage-gated proton channel from Clonorchis sinesis with a pH-sensitive fluorescent protein Super Ecliptic pHluorin A, or SEA Jin et al.
To demonstrate proof-of-principle, Kang and Baker expressed Pado in HEK cells, then used the whole-cell patch-clamp technique to depolarize one cell. The change in voltage opened the voltage-gated proton channels, facilitating the efflux of protons from the cell and creating an electrochemical gradient between this cell and neighboring cells connected via gap junctions. Protons then diffused from the neighboring cells down this electrochemical gradient, and the change in SEA fluorescence was detected in both the clamped cell and the adjacent cells.
While this method is promising, the data should be taken carefully and some calibrations allowing for quantitative analysis should be performed. A similar strategy utilizing a hybrid calcium indicator Calcium Green FlAsH could also enable detection of gap junctional couplings, by monitoring the intercellular propagation of calcium waves in gap junction coupled cells Tour et al.
Given the electrical properties of gap junctions, optogenetics is yet another useful tool for mapping gap junctions, as an electrical signal generated by light-activated channelrhodopsins Nagel et al. Recently, Wang et al. Applying blue laser illumination to the mIPNs induced depolarization of some mPNs; this effect was not altered by the nicotinic receptor antagonist mecamylamine but was sensitive to the shakB 2 mutation which affects innexin-8 Thomas and Wyman, ; Phelan et al.
Moreover, unlike the dual-electrode whole-cell patch-clamp technique, the ChIEF-based method is unidirectional and cannot be used to identify rectifying gap junctions. Compared to previous methods, these two strategies exemplified by Pado and ChIEF do not require an exogenously applied substrate, which simplifies the experimental protocol and makes them more feasible for use in in vivo applications.
In addition, because they have relatively faster kinetics on the order of milliseconds to seconds , these methods can be used to collect repeated measurements, which is essential for studying the dynamics of the strength of gap junctional connections at high temporal resolution. On the other hand, these approaches require the use of glass micropipettes, reducing their throughput. Moreover, one needs to block chemical synapses when using ChIEF to detect electrical synapses, which may alter the normal state of the nervous system.
Gap junctions play an extremely important role in mediating cell-cell communication, and their distribution and dynamics are essential for maintaining normal physiological function and homeostasis. Although researchers have been able to link genetic mutations with these conditions, identifying precisely which cell populations are affected by these mutations has been far more difficult.
In a more physiological context, single-cell transcriptomics has revealed that both neurons and glia are more heterogeneous than previously believed Lake et al. In addition, connexins and innexins are encoded by multiple genes, giving rise to a wide diversity of gap junctions. For example, the mouse and human genomes contain 20 and 21 connexin-encoding genes, respectively Sohl and Willecke, , and the Caenorhabditis elegans and Drosophila melanogaster genomes contain 25 and 8 innexin-coding genes, respectively Starich et al.
Therefore, investigating the function of gap junctions in distinct cell types and in an isoform-specific manner remains extremely challenging. To overcome these challenges, new methods providing improved genetic specificity, high spatial resolution, and functionally relevant temporal resolution are urgently needed. Ideally, these methods should be non-invasive and technically simple to perform, thereby facilitating their use in in vivo applications, allowing researchers to study gap junctions in a more physiological setting.
In principle, using genetically encoded tools provides a possible solution. On the other hand, the patch-clamp—based Pado and ChIEF strategies provide faster kinetics and do not require an exogenous substrate, making the background signal easier to control by regulating the expression level Wang et al. However, each of these methods includes a non-genetically encoded component e.
The vast majority of genetically encoded methods used to date are based on the diffusion of target molecules such as esters, ions, peptides, or synthetic dyes through gap junctions. In each case, the electrochemical gradient that drives this diffusion is generated exogenously e. In a system comprised exclusively of genetically encoded optogenetics-based components, both the generator and the reporter would be proteins e. This non-invasive optogenetics-based system could be used to control and image a large number of cells simultaneously, and the background fluorescence could be minimized greatly by controlling the expression of the generator and reporter.
Given the wide range of clear benefits associated with this approach, genetically encoded optogenetics represents one of the most promising strategies for studying gap junctions in the future. A proposed ideal optogenetics-based system for mapping gap junctions. A The principle behind the proposed optogenetics-based system shown on the left with its theoretical performance index on the right, similar to Figure 1.
B A proposed multiplex, optogenetic system for mapping gap junction using two pairs of bio-orthogonal generators and reporters and its application in an intact tissue with heterogeneous cell types. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Multiple proteins have been reported to interact or only colocalize with gap junction proteins. Squecco et al. Gap junctions are formed through cell—cell contact and homophilic cadherin—cadherin interactions. Claudin-1 colocalizes with Cx32 in rat hepatocytes lines, 71 and claudin-5 coprecipitates with Cx43 in porcine blood-brain barrier endothelial cells. Moreover, this interaction has been shown to be essential for gap junction formation. According to Govindarajan et al.
Some membrane channels and enzymes are also connexin partners. Sodium channel complexes interact with Cx43 in ventricular myocytes. The C-terminus of several connexins e. Conceivably, the interactions between Cxs and other proteins have important functions under physiological conditions and in associated diseases, such as oral disease. Tooth development depends on the sequential and reciprocal interactions between the epithelium and mesenchyme.
In the developing tooth germ of neonatal rats, Cx43 is distributed both in epithelial and mesenchymal dental cells; 82 Cx43 has been detected between ameloblasts and between odontoblasts. Once tooth germ development is completed, consistent expression of Cx43 at high levels in human dental follicle cells HDFCs is essential for tooth eruption. Cx43 plays a critical role in the development of maxillofacial structures.
During this period, Cx43 is considered to contribute to the branching morphogenesis 92 and the contractile function of myoepithelial cells, while Cx32 expression may correspond to an increase and decrease in the number of proacinar and mature acinar cells, respectively. The periodontal ligament PDL is a soft connective tissue that resides between the alveolar bone and tooth to sustain teeth and preserve tissue homoeostasis.
Periodontal tissue homoeostasis depends on a complicated cellular network that transmits signals between periodontal ligament fibroblasts PDLFs. Those proteins have two main functions. On the other hand, Cx40 and Cx45 are considered to relate to the contractile function of PDLFs, which may relate to tooth eruption.
In the rat gingival epithelia, Cx43 was detected in the basal layer and middle of the prickle cell layer. Interestingly, in the human gingival epithelia, Cx43 expression showed a progressive decrease from the spinous layers of the oral gingival epithelium to the sulcular epithelium and parts of the junctional epithelium.
Dental pulp contains fibroblasts, odontoblasts and undifferentiated mesenchymal cells, and dental pulp fibroblasts DPFs are major components among these cells. In cultured human pulp cells, Cx43 expression is upregulated during mineralization processes, indicating that Cx43 might play a role in mineralization.
Cx43 is a marker of the viability of dental pulp tissue, as Cx43 expression is reduced in aged human dental pulp. A reduction in Cx43 expression may be one characteristic of aged pulp. Connexins may be associated with cell growth, since the absence of GJIC can result in an accumulation of growth factors in cells and a suppression of contact inhibition, which together lead to cell proliferation. In the dysplasia-free oral mucosa, Cx43 is mainly expressed on the membrane in the stratum spinosum and stratum granulosum, but is not expressed in the stratum corneum.
Therefore, membrane Cx43 levels might be an independent biomarker for early changes associated with oral squamous cell carcinoma. In another study, the downregulation of Cx43 and Cx32 expression was observed in keratocystic odontogenic tumours, one of the most frequently occurring types of benign odontogenic tumours. For example, all- trans retinoic acid was shown to be beneficial for OSCC cells to regain cell—cell communication by increasing Cx32 and Cx43 expression.
Functional studies have begun to identify some of the underlying mechanisms by which connexin channel mutations contribute to oral cavity diseases. Keratitis—ichthyosis—deafness KID syndrome is a rare ectodermal dysplasia caused by mutations in the GJB2 gene, which is responsible for the production of the Cx26 protein, a protein present in the epithelial gap junctions that is postulated to be associated with the differentiation of ectodermally derived tissues.
Phenotypic features associated with Cx26 mutations are significant visual and auditory impairments. Affected patients are also at increased risk of developing epithelial malignancies.
Concerning the teeth, microdontia is present in one-fifth of the patients. More frequently, patients suffer from the amelogenesis imperfecta AI , hypoplastic type. Other dental symptoms reported include malocclusion, delayed tooth development, pulp stones, tooth loss and missing teeth.
The suppression of Cx43 expression or function promotes skin wound healing and alleviates scarring. Corresponding to this finding, Cx43 expression is substantially decreased in human gingival fibroblasts at the early stage of wound closure, and Cx43 regulates the expression of wound-healing genes in gingiva.
Thus, downregulation of Cx43 appears to be conductive to fast and scarless wound healing in gingival tissues. Increased Cx43 expression affects MMP-1 synthesis, which facilitates scar formation. Salivary glands play an important role in oral biology by secreting saliva to provide water for lubrication, as well as electrolytes, mucus, enzymes and antibacterial compounds.
Abnormal function of the salivary gland can lead to an extensive deterioration of oral health. Gap junctions have recently been suggested to be involved in maintaining salivary gland function. Therefore, an analysis of connexins and gap junctions will hopefully contribute to the study of salivary diseases. As data documenting the functions of connexins in the oral health and oral disease are still limited, information is mainly confined to the distribution of Cxs in diverse oral tissues during different developmental phases.
Far fewer reports have described the role of functional GJs in oral diseases such as periodontitis and chronic apical periodontitis. Previous studies have provided support for additional roles of Cxs in oral development and the pathogenesis and prognosis of oral diseases. Studies of various Cxs in oral tissues are categorized in Table 1.
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