Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness

Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca²⁺ levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed. connexin     

Cellular mechanisms of mutant connexins in skin disease and hearing loss

It has been demonstrated that distinct germline mutations within four connexin (Cx) genes, Cx26, Cx30, Cx31, and Cx30.3, underlie hearing loss and/or epidermal disease. Here, we describe two Cx26 mutations associated with skin disease. With the goal of understanding the mechanism(s) of Cx-associated human disease and how different mutations within the same Cx protein can result in different disorders, we performed a number of functional analyses investigating the cellular effects of disease-associated Cx mutations in keratinocytes and other cell types. Epidermal disease-associated proteins studied were primarily cytoplasmic with limited trafficking ability. FACS analysis of WT and mutant EGFP-Cx31 transfected keratinocytes revealed a high percentage of cell death associated with the skin disease-associated mutant Cx31 proteins.     

Cellular Mechanisms of Mutant Connexins in Skin Disease and Hearing Loss

It has been demonstrated that distinct germline mutations within four connexin (Cx) genes, Cx26, Cx30, Cx31, and Cx30.3, underlie hearing loss and/or epidermal disease. Here, we describe two Cx26 mutations associated with skin disease. With the goal of understanding the mechanism(s) of Cx-associated human disease and how different mutations within the same Cx protein can result in different disorders, we performed a number of functional analyses investigating the cellular effects of disease-associated Cx mutations in keratinocytes and other cell types. Epidermal disease-associated proteins studied were primarily cytoplasmic with limited trafficking ability. FACS analysis of WT and mutant EGFP-Cx31 transfected keratinocytes revealed a high percentage of cell death associated with the skin disease-associated mutant Cx31 proteins.     

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.     

Transport and Function of Cx26 Mutants Involved in Skin and Deafness Disorders

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and ΔE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and ΔE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor ΔE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and ΔE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.     

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.     

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.

     

Transport and function of cx26 mutants involved in skin and deafness disorders

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and DeltaE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and DeltaE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor DeltaE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and DeltaE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

Pathological hemichannels associated with human Cx26 mutations causing Keratitis-Ichthyosis-Deafness syndrome

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis-Ichthyosis-Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Rat Epidermal Keratinocytes as an Organotypic Model for Examining the Role of Cx43 and Cx26 in Skin Differentiation

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.     

Rat epidermal keratinocytes as an organotypic model for examining the role of Cx43 and Cx26 in skin differentiation

In order to characterize connexin expression and regulation in the epidermis, we have characterized a rat epidermal keratinocyte (REK) cell line that is phenotypically similar to basal keratinocytes in that they have the ability to differentiate into organotypic epidermis consisting of a basal cell layer, 2-3 suprabasal cell layers, and a cornified layer. RT-PCR revealed that REK cells express mRNA for Cx26, Cx31, Cx31.1, Cx37, and Cx43, which mimics the reported connexin profile for rat tissue. In addition, we report the expression of Cx30, Cx30.3, Cx40, and Cx45 in rat keratinocytes, highlighting the complexity of the connexin complement in rat epidermis. Furthermore, 3-dimensional analysis of organotypic skin revealed that Cx26 and Cx43 are exquisitely regulated during the differentiation process. The life-cycle of these connexins including their expression, transport, assembly into gap junctions, internalization, and degradation are elegantly depicted in organotypic epidermis as keratinocytes proceed from differentiation to programmed cell death.     

Connexin mutations in hearing loss,dermatological and neurological disorders

Gap junctions are important structures in cell-to-cell communication. Connexins, the protein units of gap junctions, are involved in several human disorders. Mutations in beta-connexin genes cause hearing, dermatological and peripheral nerve disorders. Recessive mutations in the gene encoding connexin 26 (GJB2) are the most common cause of childhood-onset deafness. The combination of mutations in the GJB2 and GJB6 (Cx30) genes also cause childhood hearing impairment. Although both recessive and dominant connexin mutants are functionally impaired, dominant mutations might have in addition a dominant-negative effect on wild-type connexins. Some dominant mutations in beta-connexin genes have a pleiotropic effect at the level of the skin, the auditory system and the peripheral nerves. Understanding the genotype-phenotype correlations in diseases caused by mutations in connexin genes might provide important insight into the mechanisms that lead to these disorders.     

Key functions for gap junctions in skin and hearing

Cx (connexin) proteins are components of gap junctions which are aqueous pores that allow intercellular exchange of ions and small molecules. Mutations in Cx genes are linked to a range of human disorders. In the present review we discuss mutations in β-Cx genes encoding Cx26, Cx30, Cx30.3 and Cx31 which lead to skin disease and deafness. Functional studies with Cx proteins have given insights into disease-associated mechanisms and non-gap junctional roles for Cx proteins.     

Pathological hemichannels associated with human Cx26 mutations causing Keratitis–Ichthyosis–Deafness syndrome

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis–Ichthyosis–Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Connexin 26 (GJB2) mutations as a cause of the KID syndrome with hearing loss

KID syndrome (MIM 148210) is an ectodermal dysplasia characterized by the occurrence of localized erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. KID syndrome is inherited as an autosomic dominant disease, due to mutations in the gene encoding gap junction protein GJB2 (connexin 26, Cx26). Cx26 is a component of gap junction channels in the epidermis and in the stria vascularis of the cochlea. These channels play a role in the coordinated exchange of molecules and ions occurring in a wide spectrum of cellular activities. In this paper we describe two patients with Cx26 mutations cause cell death by the alteration of protein trafficking, membrane localization and probably interfering with intracellular ion concentrations. We discuss the pathogenesis of both the hearing and skin phenotypes.     

Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.     

The effects of a mutant connexin 26 on epidermal differentiation

To elucidate the mode of action of dominant mutant connexins in causing inherited skin diseases, transgenic mice were produced that express the true Vohwinkel syndrome-associated mutant Cx26 (D66H), from a keratin 10 promoter, specifically in the suprabasal epidermal keratinocytes. Following birth, the transgenic mice developed keratoderma similar to that of human carriers of Cx26 (D66H). Expression of the transgene resulted in a loss of Cx26 and Cx30 at intercellular junctions of epidermal keratinocytes and accumulation of these connexins in the cytoplasm. Injection of primary mouse keratinocytes with Lucifer Yellow showed no difference in terms of dye spreading between transgenic and non transgenic keratinocytes in vitro. Expression of the mutant Cx26 (D66H) did not interfere with the formation of the epidermal water barrier during late embryonic development. Attempts to produce transgenic mice expressing the wild type form of Cx26 from the K10 promoter failed to produce viable animals although transgenic embryos were recovered at days 9 and 12 of gestation, suggesting that the transgene might be embryonic lethal.     

Deafness induced by Connexin 26 (GJB2) deficiency is not determined by endocochlear potential (EP) reduction but is associated with cochlear developmental disorders

Connexin 26 (Cx26, GJB2) mutations are the major cause of hereditary deafness and are responsible for >50% of nonsyndromic hearing loss. Mouse models show that Cx26 deficiency can cause congenital deafness with cochlear developmental disorders, hair cell degeneration, and the reduction of endocochlear potential (EP) and active cochlear amplification. However, the underlying deafness mechanism still remains undetermined. Our previous studies revealed that hair cell degeneration is not a primary cause of hearing loss. In this study we investigated the role of EP reduction in Cx26 deficiency-induced deafness. We found that the EP reduction is not associated with congenital deafness in Cx26 knockout (KO) mice. The threshold of auditory brainstem response (ABR) in Cx26 KO mice was even greater than 110 dB SPL, demonstrating complete hearing loss. However, the EP in Cx26 KO mice varied and not completely abolished. In some cases, the EP could still remain at higher levels (>70 mV). We further found that the deafness in Cx26 KO mice is associated with cochlear developmental disorders. Deletion of Cx26 in the cochlea before postnatal day 5 (P5) could cause congenital deafness. The cochlea had developmental disorders and the cochlear tunnel was not open. However, no congenital deafness was found when Cx26 was deleted after P5. The cochlea also displayed normal development and the cochlear tunnel was open normally. These data suggest that congenital deafness induced by Cx26 deficiency is not determined by EP reduction and may result from cochlear developmental disorders.     

Genotyping for Cx26 and Cx30 mutations in cases with congenital hearing loss

Hearing loss is the most frequent sensory defect in human being. The 13q11-q12 region contains the GJB2 and GJB6 genes, which code connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively. The 35delG, 167delT, and 235delC mutations in the Cx26 gene are the main cause for sporadic nonsyndromic hearing loss (NSHL) in many populations. The 342-kb deletion [del(GJB6-D13S1830)] of the Cx30 gene is the second most common connexin mutation after the 35delG mutation in some NSHL populations. In our study 47 hearing-impaired students were included. The Cx26 gene and the Cx30 gene were analyzed for presence of the 35delG, 167delT, and 342-kb deletion [del(GJB6-D13S1830)]. Genotyping were performed for detecting 35delG, 167delT, and del(GJB6-D13S1830) mutations using the PCR-ELISA techniques. According to the results obtained from 47 cases, the 35delG mutation was detected in 7 cases ( approximately 14.9%). Four of these mutations were determined as homozygote mutant ( approximately 8.5%), and three were determined as heterozygote mutant ( approximately 6.4%). However, 167delT and del(GJB6-D13S1830) mutations were not detected in the study group. These results support the overwhelming majority of 35delG in our study group from deafness school in our study. In conclusion, the 35delG mutation was determined as the most frequently shown mutation that leads to congenital hearing loss as in previous studies from Turkey.