The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice.

Mutations in GJB2 (Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality of Gjb2 mutations in mice, there are currently no perfect mouse models carrying Gjb2 mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygous Gjb2+/35delG and Gjb2+/235delC mutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model, Gjb235delG/35delG, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed that Gjb2 35delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.

Extracellular Vesicles and Cx43-Gap Junction Channels Are the Main Routes for Mitochondrial Transfer from Ultra-Purified Mesenchymal Stem Cells, RECs.

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ0) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ0 cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction.

Gaps and barriers: Gap junctions as a channel of communication between the soma and the germline.

Gap junctions, expressed in most tissues of the body, allow for the cytoplasmic coupling of adjacent cells and promote tissue cooperation. Gap junctions connect also the soma and the germline in many animals, and transmit somatic signals that are crucial for germline maturation and integrity. In this review, we examine the involvement of gap junctions in the relay of information between the soma and the germline, and ask whether such communication could have consequences for the progeny. While the influence of parental experiences on descendants is of great interest, the possibility that gap junctions participate in the transmission of information across generations is largely unexplored.

An Update on Connexin Gap Junction and Hemichannels in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the main causes of vision loss in the working age population. It is characterized by a progressive deterioration of the retinal microvasculature, caused by long-term metabolic alterations inherent to diabetes, leading to a progressive loss of retinal integrity and function. The mammalian retina presents an orderly layered structure that executes initial but complex visual processing and analysis. Gap junction channels (GJC) forming electrical synapses are present in each retinal layer and contribute to the communication between different cell types. In addition, connexin hemichannels (HCs) have emerged as relevant players that influence diverse physiological and pathological processes in the retina. This article highlights the impact of diabetic conditions on GJC and HCs physiology and their involvement in DR pathogenesis. Microvascular damage and concomitant loss of endothelial cells and pericytes are related to alterations in gap junction intercellular communication (GJIC) and decreased connexin 43 (Cx43) expression. On the other hand, it has been shown that the expression and activity of HCs are upregulated in DR, becoming a key element in the establishment of proinflammatory conditions that emerge during hyperglycemia. Hence, novel connexin HCs blockers or drugs to enhance GJIC are promising tools for the development of pharmacological interventions for diabetic retinopathy, and initial in vitro and in vivo studies have shown favorable results in this regard.

Mast Cell and Astrocyte Hemichannels and Their Role in Alzheimer's Disease, ALS, and Harmful Stress Conditions.

Considered relevant during allergy responses, numerous observations have also identified mast cells (MCs) as critical effectors during the progression and modulation of several neuroinflammatory conditions, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). MC granules contain a plethora of constituents, including growth factors, cytokines, chemokines, and mitogen factors. The release of these bioactive substances from MCs occurs through distinct pathways that are initiated by the activation of specific plasma membrane receptors/channels. Here, we focus on hemichannels (HCs) formed by connexins (Cxs) and pannexins (Panxs) proteins, and we described their contribution to MC degranulation in AD, ALS, and harmful stress conditions. Cx/Panx HCs are also expressed by astrocytes and are likely involved in the release of critical toxic amounts of soluble factors-such as glutamate, adenosine triphosphate (ATP), complement component 3 derivate C3a, tumor necrosis factor (TNFα), apoliprotein E (ApoE), and certain miRNAs-known to play a role in the pathogenesis of AD, ALS, and other neurodegenerative disorders. We propose that blocking HCs on MCs and glial cells offers a promising novel strategy for ameliorating the progression of neurodegenerative diseases by reducing the release of cytokines and other pro-inflammatory compounds.

Constitutive deletion of astrocytic connexins aggravates kainate-induced epilepsy.

The astroglial gap junctional network formed by connexin (Cx) channels plays a central role in regulating neuronal activity and network synchronization. However, its involvement in the development and progression of epilepsy is not yet understood. Loss of interastrocytic gap junction (GJ) coupling has been observed in the sclerotic hippocampus of patients with mesial temporal lobe epilepsy (MTLE) and in mouse models of MTLE, leading to the suggestion that it plays a causative role in the pathogenesis. To further elucidate this clinically relevant question, we investigated consequences of astrocyte disconnection on the time course and severity of kainate-induced MTLE with hippocampal sclerosis (HS) by comparing mice deficient for astrocytic Cx proteins with wild-type mice (WT). Continuous telemetric EEG recordings and video monitoring performed over a period of 4 weeks after epilepsy induction revealed substantially higher seizure and interictal spike activity during the chronic phase in Cx deficient versus WT mice, while the severity of status epilepticus was not different. Immunohistochemical analysis showed that, despite the elevated chronic seizure activity, astrocyte disconnection did not aggravate the severity of HS. Indeed, the extent of CA1 pyramidal cell loss was similar between the experimental groups, while astrogliosis, granule cell dispersion, angiogenesis, and microglia activation were even reduced in Cx deficient as compared to WT mice. Interestingly, seizure-induced neurogenesis in the adult dentate gyrus was also independent of astrocytic Cxs. Together, our data indicate that constitutive loss of GJ coupling between astrocytes promotes neuronal hyperexcitability and attenuates seizure-induced histopathological outcomes.

Role of ROS/RNS in Preeclampsia: Are Connexins the Missing Piece?

Preeclampsia is a pregnancy complication that appears after 20 weeks of gestation and is characterized by hypertension and proteinuria, affecting both mother and offspring. The cellular and molecular mechanisms that cause the development of preeclampsia are poorly understood. An important feature of preeclampsia is an increase in oxygen and nitrogen derived free radicals (reactive oxygen species/reactive nitrogen species (ROS/RNS), which seem to be central players setting the development and progression of preeclampsia. Cell-to-cell communication may be disrupted as well. Connexins (Cxs), a family of transmembrane proteins that form hemichannels and gap junction channels (GJCs), are essential in paracrine and autocrine cell communication, allowing the movement of signaling molecules between cells as well as between the cytoplasm and the extracellular media. GJCs and hemichannels are fundamental for communication between endothelial and smooth muscle cells and, therefore, in the control of vascular contraction and relaxation. In systemic vasculature, the activity of GJCs and hemichannels is modulated by ROS and RNS. Cxs participate in the development of the placenta and are expressed in placental vasculature. However, it is unknown whether Cxs are modulated by ROS/RNS in the placenta, or whether this potential modulation contributes to the pathogenesis of preeclampsia. Our review addresses the possible role of Cxs in preeclampsia, and the plausible modulation of Cxs-formed channels by ROS and RNS. We suggest these factors may contribute to the development of preeclampsia.

Bursting at the Seams: Molecular Mechanisms Mediating Astrocyte Swelling.

Brain swelling is one of the most robust predictors of outcome following brain injury, including ischemic, traumatic, hemorrhagic, metabolic or other injury. Depending on the specific type of insult, brain swelling can arise from the combined space-occupying effects of extravasated blood, extracellular edema fluid, cellular swelling, vascular engorgement and hydrocephalus. Of these, arguably the least well appreciated is cellular swelling. Here, we explore current knowledge regarding swelling of astrocytes, the most abundant cell type in the brain, and the one most likely to contribute to pathological brain swelling. We review the major molecular mechanisms identified to date that contribute to or mitigate astrocyte swelling via ion transport, and we touch upon the implications of astrocyte swelling in health and disease.

Accessing gap-junction channel structure-function relationships through molecular modeling and simulations.

BACKGROUND: Gap junction channels (GJCs) are massive protein channels connecting the cytoplasm of adjacent cells. These channels allow intercellular transfer of molecules up to ~1 kDa, including water, ions and other metabolites. Unveiling structure-function relationships coded into the molecular architecture of these channels is necessary to gain insight on their vast biological function including electrical synapse, inflammation, development and tissular homeostasis. From early works, computational methods have been critical to analyze and interpret experimental observations. Upon the availability of crystallographic structures, molecular modeling and simulations have become a valuable tool to assess structure-function relationships in GJCs. Modeling different connexin isoforms, simulating the transport process, and exploring molecular variants, have provided new hypotheses and out-of-the-box approaches to the study of these important channels. METHODS: Here, we review foundational structural studies and recent developments on GJCs using molecular modeling and simulation techniques, highlighting the methods and the cross-talk with experimental evidence. RESULTS AND DISCUSSION: By comparing results obtained by molecular modeling and simulations techniques with structural and functional information obtained from both recent literature and structural databases, we provide a critical assesment of structure-function relationships that can be obtained from the junction between theoretical and experimental evidence.

Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness.

Gap-junction channels (GJCs) are formed by head-to-head association of two hemichannels (HCs, connexin hexamers). HCs and GJCs are permeable to ions and hydrophilic molecules of up to Mr ~1 kDa. Hearing impairment of genetic origin is common, and mutations of connexin 26 (Cx26) are its major cause. We recently identified two novel Cx26 mutations in hearing-impaired subjects, L10P and G109V. L10P forms functional GJCs with slightly altered voltage dependence and HCs with decrease ATP/cationic dye selectivity. G109V does not form functional GJCs, but forms functional HCs with enhanced extracellular Ca(2+) sensitivity and subtle alterations in voltage dependence and ATP/cationic dye selectivity. Deafness associated with G109V could result from decreased GJCs activity, whereas deafness associated to L10P may have a more complex mechanism that involves changes in HC permeability.

Connexinopathies: a structural and functional glimpse.

Mutations in human connexin (Cx) genes have been related to diseases, which we termed connexinopathies. Such hereditary disorders include nonsyndromic or syndromic deafness (Cx26, Cx30), Charcot Marie Tooth disease (Cx32), occulodentodigital dysplasia and cardiopathies (Cx43), and cataracts (Cx46, Cx50). Despite the clinical phenotypes of connexinopathies have been well documented, their pathogenic molecular determinants remain elusive. The purpose of this work is to identify common/uncommon patterns in channels function among Cx mutations linked to human diseases. To this end, we compiled and discussed the effect of mutations associated to Cx26, Cx32, Cx43, and Cx50 over gap junction channels and hemichannels, highlighting the function of the structural channel domains in which mutations are located and their possible role affecting oligomerization, gating and perm/selectivity processes.

Carbon monoxide: A new player in the redox regulation of connexin hemichannels.

Carbon monoxide (CO) is a gaseous transmitter that is known to be involved in several physiological processes, but surprisingly it is also becoming a promising molecule to treat several pathologies including stroke and cancer. CO can cross the plasma membrane and activate guanylate cyclase, increasing the cGMP concentration and activating some kinases, including PKG. The other mechanism of action involves induction of protein carbonylation. CO is known to directly and indirectly modulate the function of ion channels at the plasma membrane, which in turn have important repercussions in the cellular behavior. One group of these channels is hemichannels, which are formed by proteins known as connexins (Cxs). Hemichannel allows not only the flow of ions through their pore but also the release of molecules such as ATP and glutamate. Therefore, their modulation not only impacts cellular function but also cellular communication, having the capability to affect tissular behavior. Here, we review the most recent results regarding the effect of CO on Cx hemichannels and their possible repercussions on pathologies.

Connexin 36 mediates blood cell flow in mouse pancreatic islets.

The insulin-secreting ß-cells are contained within islets of Langerhans, which are highly vascularized. Blood cell flow rates through islets are glucose-dependent, even though there are no changes in blood cell flow within in the surrounding exocrine pancreas. This suggests a specific mechanism of glucose-regulated blood flow in the islet. Pancreatic islets respond to elevated glucose with synchronous pulses of electrical activity and insulin secretion across all ß-cells in the islet. Connexin 36 (Cx36) gap junctions between islet ß-cells mediate this synchronization, which is lost in Cx36 knockout mice (Cx36(-/-)). This leads to glucose intolerance in these mice, despite normal plasma insulin levels and insulin sensitivity. Thus, we sought to investigate whether the glucose-dependent changes in intraislet blood cell flow are also dependent on coordinated pulsatile electrical activity. We visualized and quantified blood cell flow using high-speed in vivo fluorescence imaging of labeled red blood cells and plasma. With the use of a live animal glucose clamp, blood cell flow was measured during either hypoglycemia (∼50 mg/dl) or hyperglycemia (∼300 mg/dl). In contrast to the large glucose-dependent islet blood velocity changes observed in wild-type mice, only minimal differences are observed in both Cx36(+/-) and Cx36(-/-) mice. This observation supports a novel model where intraislet blood cell flow is regulated by the coordinated electrical activity in the islet ß-cells. Because Cx36 expression and function is reduced in type 2 diabetes, the resulting defect in intraislet blood cell flow regulation may also play a significant role in diabetic pathology.

Inhibition of connexin 36 hemichannels by glucose contributes to the stimulation of insulin secretion.

The existence of functional connexin36 (Cx36) hemichannels in ß-cells was investigated in pancreatic islets of rat and wild-type (Cx36(+/+)), monoallelic (Cx36(+/-)), and biallelic (Cx36(-/-)) knockout mice. Hemichannel opening by KCl depolarization was studied by measuring ATP release and changes of intracellular ATP (ADP). Cx36(+/+) islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36(-/-) than Cx36(+/+) islets and was not decreased by KCl depolarization; Cx36(+/-) islets showed values between that of control and homozygous islets. Five minimolar extracellular ATP increased ATP content and ATP/ADP ratio and induced a biphasic insulin secretion in depolarized Cx36(+/+) and Cx36(+/-) but not Cx36(-/-) islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential, and their activation was inhibited by mefloquine and glucose (IC50 ∼8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet ß-cells might avoid depolarization-induced ATP loss, allowing an optimum increase of the ATP/ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36(+/-) and Cx36(-/-) islets confirms that Cx36 gap junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.

Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model.

BACKGROUND: Extravillous trophoblasts (EVTs) form stratified columns at the placenta-uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. METHODS: We developed two cellular models, one with "low Cx40" (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with "high Cx40" (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. CONCLUSIONS: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NO.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Protector Role of Cx30.2 in Pancreatic ß-Cell against Glucotoxicity-Induced Apoptosis.

Glucotoxicity may exert its deleterious effects on pancreatic ß-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. ß-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in ß cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of ß cells (RIN-m5F). METHODS: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. RESULTS: High glucose did not induce apoptosis in RIN-m5F ß cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. CONCLUSION: The data suggest that the upregulation of Cx30.2 protects ß cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

Connexins in Cancer, the Possible Role of Connexin46 as a Cancer Stem Cell-Determining Protein.

Cancer is a widespread and incurable disease caused by genetic mutations, leading to uncontrolled cell proliferation and metastasis. Connexins (Cx) are transmembrane proteins that facilitate intercellular communication via hemichannels and gap junction channels. Among them, Cx46 is found mostly in the eye lens. However, in pathological conditions, Cx46 has been observed in various types of cancers, such as glioblastoma, melanoma, and breast cancer. It has been demonstrated that elevated Cx46 levels in breast cancer contribute to cellular resistance to hypoxia, and it is an enhancer of cancer aggressiveness supporting a pro-tumoral role. Accordingly, Cx46 is associated with an increase in cancer stem cell phenotype. These cells display radio- and chemoresistance, high proliferative abilities, self-renewal, and differentiation capacities. This review aims to consolidate the knowledge of the relationship between Cx46, its role in forming hemichannels and gap junctions, and its connection with cancer and cancer stem cells.

"Cytokine-microfactories" recruit DCs and deliver tumor antigens via gap junctions for immunotherapy.

The majority (~80%) of patients with cancer do not derive clinical benefit from current immunotherapy, largely due to attenuation of immune responses imposed by robust immunosuppression at tumor sites. Here, a cell-based tumor antigen delivery strategy was developed to boost tumor-specific immunity. Notably, the platform constructing ferric oxide nanoparticle-trained macrophages loading tumor antigens (MFe-N) acquired an immunostimulatory program and functioned as the tumoritropic "cytokine-microfactories" to sustainably produce high levels of multiple therapeutic cytokines (GM-CSF, TNFα, and MIP-1α), which are important in activation of immune cells with antitumor potential. Indeed, MFe-N markedly enhanced recruitment of the professional antigen-presenting cells, dendritic cells (DCs), to the tumor sites of an established B16F10 mouse melanoma model. Subsequently, MFe-N effectively delivered tumor antigens to DCs by gap junction-mediated cell-to-cell transmission. And this trafficking was critical for DC maturation to augment antitumor T-cell responses. Simultaneously, the "cytokine-microfactories" elicited high production of the tumoricidal effectors, and in turn blunted the pro-angiogenic activity of tumor-associated macrophages, resulting in conversion of the tumor-supporting milieu to a tumoricidal function that favored infiltration of antitumor T-cells. The findings provided a novel "cytokine-microfactories" harnessing effective delivery of tumor antigens and production of therapeutic cytokines to robustly promote antigen presentation and reshape the tumor immune milieu for priming antitumor immunity. This can enhance existing T-cell mediated immunotherapeutic potency and extend the curative potential immunotherapy to a broader range of patients.

Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the GJB2 (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss.

Mutations in the GJB2 gene encoding transmembrane protein connexin 26 (Cx26) are the most common cause for hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3) deafness and syndromes associating hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare GJB2 variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of tryptophan at a conserved amino acid position 172 with cysteine (p.Trp172Cys) in the second extracellular loop of Cx26 protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys protein by immunocytochemistry and the hemichannels permeability by dye loading assay. The GJB2 knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different GJB2 variants (wild type and mutations associated with hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26 protein in pathogenesis of deafness.