Keratitis-ichthyosis-deafness Syndrome Research Articles

The D50N mutation and syndromic deafness: Altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions

Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extracellular Ca2+, decreased unitary conductance, increased open hemichannel current rectification and voltage-shifted activation. We demonstrate that D50 is a pore-lining residue and that negative charge at this position strongly influences open hemichannel properties. Examination of two putative intersubunit interactions involving D50 suggested by the Cx26 crystal structure, K61–D50 and Q48–D50, showed no evidence of a K61–D50 interaction in hemichannels. However, our data suggest that Q48 and D50 interact and disruption of this interaction shifts hemichannel activation positive along the voltage axis. Additional shifts in activation by extracellular Ca2+ remained in the absence of a D50–Q48 interaction but required an Asp or Glu at position 50, suggesting a separate electrostatic mechanism that critically involves this position. In gap junction (GJ) channels, D50 substitutions produced loss of function, whereas K61 substitutions functioned as GJ channels but not as hemichannels. These data demonstrate that D50 exerts effects on Cx26 hemichannel and GJ channel function as a result of its dual role as a pore residue and a component of an intersubunit complex in the extracellular region of the hemichannel. Differences in the effects of substitutions in GJ channels and hemichannels suggest that perturbations in structure occur upon hemichannel docking that significantly impact function. Collectively, these data provide insight into Cx26 structure–function and the underlying bases for the phenotypes associated with KID syndrome patients carrying the D50N mutation.

Conserved glycine at position 45 of major cochlear connexins constitutes a vital component of the Ca2+ sensor for gating of gap junction hemichannels

Mutations in gap junction (GJ) family of proteins, especially in the connexin (Cx) 26, are responsible for causing severe congenital hearing loss in a significant portion of patients (30–50% in various ethnic groups). Substitution of glycine at the position 45 of Cx26 to glutamic acid (p.G45E mutation) causes the Keratitis–ichthyosis–deafness (KID) syndrome. Previous studies have suggested that this point mutation caused a gain-of-function defect. However, the molecular mechanism of KID syndrome remains unclear. Since glycine at this position is conserved in many Cxs expressed in the cochlea, we tested the hypothesis that glycine at position 45 is an important component of the sensor regulating the Ca2+ gating of GJ hemichannels. Using reconstituted Cx30, 32 and 43 expressed in the HEK 293 cells, we compared the functions of wild type and p.G45E mutant Cxs. We found that G45E in Cx30 resulted in similar deleterious cellular effects as Cx26 did. Cell death occurred within 24h of transfection, which was rescued by increasing extracellular Ca2+ concentration ([Ca2+]o). Dye loading assay showed that Cx30 G45E, similar to Cx26 G45E, had leaky hemichannels at physiological [Ca2+]o (1.2mM). Higher [Ca2+]o reduced the dye loading in a dose-dependent manner. Whole cell membrane current recordings also indicated that G45E caused increased hemichannel activities. p.G45E mutations of Cx32 and 43 also resulted in leaky hemichannels compared to their respective wild types in lower [Ca2+]o. Our data in this study provided further support for the hypothesis that glycine at position 45 is a conserved Ca2+ sensor for the gating of GJ hemichannels among multiple Cx subtypes expressed in the cochlea.

The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

Squamous Cell Carcinoma Arising from Keratitis–Ichthyosis–Deafness Syndrome

Inherited ichthyoses are rare genodermatoses caused by mutations in the genes involved in epidermal develop-ment. Keratitis–ichthyosis–deafness (KID) syndrome is a debilitating ectodermal dysplasia that predisposes patients to develop squamous cell carcinomas (SCC) in addition to leading to profound sensory deafness and erythrokeratoderma already present at birth (1–5). Re-cent reports have provided evidence that KID syndrome is caused by a mutation of connexin 26 (Cx26) (2–5), a gap junction protein, encoded by GJB2, with the ma-jority of patients harbouring the D50N mutation (2–8). Interestingly, a previous review suggested that the mis-sense D50N mutation in patients with KID syndrome is strongly connected with the development of skin SCC (1), although not every case develops malignancies (6, 7). We describe here a case of large areas of SCC arising from KID syndrome after severe infection. Our report also suggests that severe bacterial infection might be one of the reasons for the establishment of this aggres-sive skin cancer. CaSe RepORt

Dental Treatments under the General Anesthesia in a Child with Keratitis, Ichthyosis, and Deafness Syndrome.

KID syndrome is a rare genodermatosis characterized by keratitis, ichthyosis, and sensorineural deafness. Although the dermatological, ophthalmologic, and sensorineural defects are emphasized in the literature, oral and dental evaluations are so superficial. In this case report, dental and oral symptoms of a three year and five months old boy with KID syndrome, suffering severe Early Childhood Caries (s-ECC) and dental treatments done under General Anesthesia (GA) were reported.

Diseases associated with hidranitis suppurativa: part 2 of a series on hidradenitis

Hidradenitis suppurativa (HS), a pathologic follicular disease, impacts patients' lives profoundly and usually occurs in isolation. The diseases with the strongest association are obesity, depression, and pain. HS is associated with many diseases including acne conglobata (AC), dissecting cellulitis, pilonidal cysts, and obesity. Pyoderma fistulans sinifica (fox den disease) appears to be the same entity as Hurley Stage 2 of 3 HS. The rate of acne vulgaris in HS patients mirrors unaffected controls. The most common, albeit still uncommon, association is with seronegative, haplotype unlinked arthritis (most importantly B27), in particular spondolyarthritis. Crohn disease and HS occur together at a rate that varies from 0.6% to 38% in retrospective cases series. Ulcerative colitis occurred with HS in 14% of patients in one series. The next most common association is with pyoderma gangrenosum, but this association is likely under-reported. Synovitis-Acne-Pustulosis Hyperostosis-Osteitis (SAPHO) syndrome, which is rare, has more than 10 reports linking it to HS. Nine case reports have linked Dowling-Degos disease (DDD) to HS and two reports related HS to Fox-Fordyce disease (FF), but because both occur in the axilla this might be a mere coincidence. HS is rarely associated with ophthalmic pathology. Specifically, more than 5 reports link it to Keratitis-Ichthyosis-Deafness syndrome (KID); greater than10 cases link it to interstitial keratitis and 2 cases are linked to Behçet's disease. The presence of proteinuria and acute nephritis link HS to the kidney, especially since and reports have documented resolution of HS after renal transplant. Florid steatocystoma multiplex, Sjogren Syndrome, and HS have been linked and their reports likely underestimate their coincidence because all these entities involve occlusion (albeit by different mechanisms). Three reports link HS and amyloid, but both share some common genetic underpinnings and thus the coincidence of these diseases is likely underreported. Pyoderma vegetans has been noted in 2 cases of HS and 4 cases of Inflammatory Bowel Disease (IBD) and is likely a clue to the linkage of the pathology of IBD and HS. Pityriasis rubra pilaris, in particular Type VI related to HIV, has a relationship more commonly with acne conglobata, but with HS also. Single case reports of diseases associated with HS include systemic lupus erythematosus, acromegaly, Down syndrome, Bazex-Dupre´-Christol, and prurtis ani, but these might be coincidences. Pyogenic Arthritis, Pyoderma gangrenosum, and Acne (PAPA Syndrome) and Pyoderma gangrenosum, Acne, and Suppurative Hidradenitis (PASH Syndrome) are pyodermic-arthritic syndromes that are associated with HS. Erythema nodosum and granulomatous lobular mastitis have been reported with HS but the significance of these reports is uncertain. Because of scarring, HS can result in lymphedema including scrotal elephantiasis and verrucous lymphedema. HS is sometimes accompanied by obesity, hypertension, and anemia and can be considered a disease in the spectrum of metabolic syndrome, a skin disease with systemic consequences. HS, like other types of chronic inflammation when long standing in the perianal and perineal areas, can result in squamous cell cancer. A variety of drugs can induce HS. These include lithium, sirolimus, cyclosporine, vemurafenib, and oral contraceptives. Inverse psoriasis or psoriasis vulgaris as a side effect of infliximab therapy may be associated with HS. These associations aside, most cases of HS occur in isolation without coincident morbidity.

Porokeratotic Eccrine Nevus May Be Caused by Somatic Connexin26 Mutations

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis–ichthyosis–deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.

Mutation analysis of GJB2 gene in a patient with keratitis-ichthyosis-deafness syndrome

Objective To investigate the clinical features of and GJB2 gene mutations in a Chinese Han patient with keratitis-ichthyosis-deafness syndrome (KID syndrome),in hope to offer evidence for the clinical and genetic diagnosis of KID syndrome.Methods Clinical data were collected from a patient with KID syndrome.DNA was extracted from peripheral blood of the patient and his two family members (mother and brother).PCR was performed to amplify the exon 2 and its flanking splicing sites of GJB2 gene followed by bidirectional direct DNA sequencing. Results The patient presented with the typical triad of vascularizing keratitis,ichthyosis and congenital deafness.A G148A mutation in the exon 2 of GJB2 gene,resulting in the substitution of aspartic acid by asparagine at position 50 of the junction protein connexin 26 (Cx26),was identified in the patient,but not in either of his family members.Conclusion The G148A mutation in GJB2 gene may be responsible for the clinical phenotype of KID syndrome in this Chinese patient.

Prevention of Floppy Kid Syndrome: A long-term clinical field study conducted on a goat farm in South Tyrol/Italy

ObjectiveTo discover whether Floppy Kid Syndrome can be prevented by separating kids at birth from their dams and rearing them on bovine colostrum followed by cows’ milk. DesignClinical field study during the period of 2000–2010. Materials and methodsThe study was conducted on a goat farm in South Tyrol, Italy. Kids were separated at birth from their dams and reared on bovine colostrum followed by cows’ milk (group 1); kids were also reared traditionally leaving them to suckle with their respective dams (group 2). In both groups, clinical symptoms were recorded. In those exhibiting FKS, associated symptoms and recovery after treatment were documented. Cumulative probability of survival and determination of hazard rate were plotted using the Kaplan–Meier method. ResultsA total of 414 kids born on this farm over this period (2000–2010) were examined. None of the 146 kids of group 1 acquired FKS. In group 2, 16 from the 268 kids reared traditionally developed typical FKS. The symptoms of FKS, which were observed included altered general condition (apathy, somnolence, and lethargy), altered posture (knuckling over in the carpal joints, ataxia, and recumbency), salivation and abomasal distension. The difference between the groups was significant (χ2 test, p=0.003). Distribution of FKS in group 2 revealed that in January and February no cases of FKS occurred. In March, April, and May morbidity was at 2.6%, 69.2%, and 71.4%, respectively. Survival analysis in group 1 revealed a cumulative probability of survival of 1.00 and a hazard rate of 0.00 during the whole observation period (January to May). In group 2, a cumulative probability of survival of 1.00 for the first two days after birth, declining to 0.94 on the 11th day, was demonstrated. The difference in time between the groups was significant (p=0.003) applying the Log Rank test. In group 2, for the period of March to May survival analysis revealed a cumulative probability of survival of 1.00 for the first day after birth, declining to 0.84 on the 11th day. This indicates a morbidity rate of 16% for this period. On the 4th and 5th day post partum the hazard rate was highest (HR=0.04). After 11 days neither cumulative probability of survival nor hazard rate declined. The results of this study demonstrate an 100% probability that FKS will not affect kids after 11 days of age. A positive correlation was apparent between the recorded outdoor temperature and the incidence of Floppy Kid Syndrome. Conclusions and clinical relevanceIn this long-term study conducted on a goat farm in South Tyrol Floppy Kid Syndrome in goat kids can be prevented by separating neonate kids from the dams, and rearing the kids with bovine colostrum/milk. The important factor seems to be the separation of new born kids from adults, thus preventing exposure of kids via sucking/licking to the bacterial agent causing d-lactic acidosis, originating from the adult goats or their immediate environment. Greatest susceptibility for Floppy Kid Syndrome is in the period from March to May between the 3rd and 6th day post partum.

Connexin 26 (GJB2) mutations in keratitis–ichthyosis–deafness syndrome presenting with squamous cell carcinoma

Connexin 26 (<i>GJB2</i>) mutations in keratitis–ichthyosis–deafness syndrome presenting with squamous cell carcinoma

KID syndrome: histopathological, immunohistochemical and molecular analysis of precancerous and cancerous skin lesions

KID syndrome: histopathological, immunohistochemical and molecular analysis of precancerous and cancerous skin lesions

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

Extending the Phenotypic Spectrum of Keratitis‐Ichthyosis‐Deafness Syndrome: Report of a Patient with GJB2 (G12R) Connexin 26 Mutation and Unusual Clinical Findings

Keratitis-ichthyosis-deafness (KID) syndrome is a rare ectodermal dysplasia, characterized mainly by the presence of hyperkeratotic skin lesions, neurosensory hearing loss, and vascularizing keratitis. Most mutations that have been discovered as a cause of KID syndrome are autosomal dominant, found in exon 2 of the Connexin (Cx) 26 gene. A G12R (p.Gly12Arg) is a GJB2 mutation reported in only two patients with KID syndrome to date. This article describes a patient with the G12R mutation and KID syndrome with interesting additional features, which include a porokeratotic eccrine ostial and dermal duct nevus, follicular occlusion triad, and unusual persistent oral mucosal papules. We compare this patient's phenotype with the only two other patients described with the same (G12R) mutation. The phenotypic heterogeneity of KID syndrome, inexplicable according to our current understanding of these proteins, speaks to the complexity of the connexin system and its overlapping expression patterns in different tissues.

The Changes of Auditory Performance After Cochlear Implantation in Child with KID syndrome

The Changes of Auditory Performance After Cochlear Implantation in Child with KID syndrome

Connexin 26 ( GJB2) mutation in KID syndrome: An Egyptian patient

Keratitis ± ichthyosis ± deafness (KID) syndrome is a rare disorder characterized by the occurrence of localized erythematous scaly skin lesions, severe bilateral keratitis, and sensorineural deafness. Other ocular manifestations include corneal epithelial defects and scarring, which cause progressive decline of visual acuity and may eventually lead to blindness. To our knowledge, few cases have been reported worldwide and none were reported from the Middle East Arab countries. Here we report the first Egyptian patient with this syndrome.

Molecular analysis of connexin26 asparagine14 mutations associated with syndromic skin phenotypes

Mutations in connexin26, a cutaneous gap junction protein, cause a wide variety of skin disorders including keratitis-ichthyosis-deafness syndrome (KID). We previously delineated a phenotype distinct from KID, hypotrichosis-deafness syndrome, caused by the mutation p.Asn14Lys in connexin26. However, a different mutation at the same location, p.Asn14Tyr, was reported to cause a disorder similar to KID. Distinct substitutions cause different conformational changes to the protein, each with unique consequences for its behaviour. This may explain the phenotypic differences. We found the previously described mutation p.Asn14Tyr in connexin26 in two patients from Brazil and Poland, and observe quite distinct phenotypes distinguishable from classical KID syndrome. We assessed functional consequences of p.Asn14Tyr and p.Asn14Lys, using fluorescently labelled proteins and parachute assay, comparing them with the classical KID mutation p.Asp50Asn. Our analyses show that p.Asn14Tyr, p.Asn14Lys and p.Asp50Asn have different consequences for protein localization and gap junction permeability. However, the differences between the phenotypes we observed cannot be readily explained from effects on protein trafficking or gap junction permeability.

A novel mutation in the connexin 26 gene (GJB2) in a child with clinical and histological features of keratitis–ichthyosis–deafness (KID) syndrome

Keratitis-ichthyosis-deafness (KID) syndrome is a rare congenital ectodermal disorder, caused by heterozygous missense mutation in GJB2, encoding the gap junction protein connexin 26. The commonest mutation is the p.Asp50Asn mutation, and only a few other mutations have been described to date. To report the fatal clinical course and characterize the genetic background of a premature male neonate with the clinical and histological features of KID syndrome. Genomic DNA was extracted from peripheral blood and used for PCR amplification of the GJB2 gene. Direct sequencing was used for mutation analysis. The clinical features included hearing impairment, ichthyosiform erythroderma with hyperkeratotic plaques, palmoplantar keratoderma, alopecia of the scalp and eyelashes, and a thick vernix caseosa-like covering of the scalp. On histological analysis, features characteristic of KID syndrome, such as acanthosis and papillomatosis of the epidermis with basket-weave hyperkeratosis, were seen. The skin symptoms were treated successfully with acitretin 0.5 mg/kg. The boy developed intraventricular and intracerebral haemorrhage, leading to hydrocephalus. His condition was further complicated by septicaemia and meningitis caused by infection with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae. Severe respiratory failure followed, and the child died at 46 weeks of gestational age (13 weeks postnatally). Sequencing of the GJB2 gene showed that the child was heterozygous for a novel nucleotide change, c.263C>T, in exon 2, leading to a substitution of alanine for valine at position 88 (p.Ala88Val). This study has identified a new heterozygous de novo mutation in the Cx26 gene (c.263C>T; p.Ala88Val) leading to KID syndrome.

The connexin26 S17F mouse mutant represents a model for the human hereditary keratitis-ichthyosis-deafness syndrome

Mutations in the GJB2 gene coding for connexin26 (Cx26) can cause a variety of deafness and hereditary hyperproliferative skin disorders in humans. In this study, we investigated the Cx26S17F mutation in mice, which had been identified to cause the keratitis-ichthyosis-deafness (KID) syndrome in humans. The KID syndrome is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia, sensorineural hearing loss and increased carcinogenic potential. We have generated a conditional mouse mutant, in which the floxed wild-type Cx26-coding DNA can be deleted and the Cx26S17F mutation is expressed under control of the endogenous Cx26 promoter. Homozygous mutants are not viable, whereas the surviving heterozygous mice show hyperplasia of tail and foot epidermis, wounded tails and annular tail restrictions, and are smaller than their wild-type littermates. Analyses of auditory brainstem responses (ABRs) indicate an ∼35 dB increased hearing threshold in these mice, which is likely due to the reduction of the endocochlear potential by 20-40%. Our results indicate that the Cx26S17F protein, which does not form functional gap junction channels or hemichannels, alters epidermal proliferation and differentiation in the heterozygous state. In the inner ear, reduced intercellular coupling by heteromeric channels composed of Cx26S17F and Cx30 could contribute to hearing impairment in heterozygous mice, while remaining wild-type Cx26 may be sufficient to stabilize Cx30 and partially maintain cochlear homeostasis. The phenotype of heterozygous mice resembles many of the symptoms of the human KID syndrome. Thus, these mice represent an appropriate model to further investigate the disease mechanism.

Aural Rehabilitation after Cochlear Implantation in a Child with Keratitis-Ichthyosis-Deafness (KID) Syndrome

Keratitis-ichthyosis-deafness (KID) syndrome is a rare ectodermal dysplasia characterized by vascularizing keratitis, profound sensorineural hearing loss (SNHL), and progressive erythrokeratoderma. It is a clinical triad that indicates a failure in development and differentiation of multiple stratifying epithelia. KID syndrome has been associated with mutations in the gene encoding connexin 26, a gap junction protein. The aim of this case review was to investigate auditory performances after cochlear implantation and emphasize the importance of auditory rehabilitation. The results of this case suggested that auditory performances and communication skills of a child with KID syndrome have improved through the cochlear implantation and aural rehabilitation. In particular listening tests of Listening Progress Profile (Lip Profile) and Meaningful Auditory Integration Scale (MAIS), the scores have improved from 52% to 97% and 20% to 80%, respectively. The results of this study suggested that auditory rehabilitation and cochlear implantation in children with (KID) syndrome can be effective and important. KEY WORDS：Aural rehabilitation·Cochlear implantation·Keratitis-ichthyosis-deafness (KID) syndrome.

Differentially altered Ca2+ regulation and Ca2+ permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome

Mutations in GJB2, which encodes Cx26, are one of the most common causes of inherited deafness in humans. More than 100 mutations have been identified scattered throughout the Cx26 protein, most of which cause nonsyndromic sensorineural deafness. In a subset of mutations, deafness is accompanied by hyperkeratotic skin disorders, which are typically severe and sometimes fatal. Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (E1) domains. Here, we examined two such mutations, A40V and G45E, which are positioned near the TM1/E1 boundary and are associated with keratitis ichthyosis deafness (KID) syndrome. Both of these mutants have been reported to form hemichannels that open aberrantly, leading to “leaky” cell membranes. Here, we quantified the Ca2+ sensitivities and examined the biophysical properties of these mutants at macroscopic and single-channel levels. We find that A40V hemichannels show significantly impaired regulation by extracellular Ca2+, increasing the likelihood of aberrant hemichannel opening as previously suggested. However, G45E hemichannels show only modest impairment in regulation by Ca2+ and instead exhibit a substantial increase in permeability to Ca2+. Using cysteine substitution and examination of accessibility to thiol-modifying reagents, we demonstrate that G45, but not A40, is a pore-lining residue. Both mutants function as cell–cell channels. The data suggest that G45E and A40V are hemichannel gain-of-function mutants that produce similar phenotypes, but by different underlying mechanisms. A40V produces leaky hemichannels, whereas G45E provides a route for excessive entry of Ca2+. These aberrant properties, alone or in combination, can severely compromise cell integrity and lead to increased cell death.