Search

Background: Members of the β-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts., Methods: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules., Results: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels., Conclusions: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions., (© 2024. The Author(s).)

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygc dup ). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygc dup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Our recent studies in mice suggest that a crucial event for the development of cataracts is the formation of calcium-containing deposits. To examine the generality of pathologic mineralization as a novel mechanism of cataract formation, we analyzed lens material from different human cataract surgeries. Human lens material was obtained from routine cataract surgeries performed on three patients with dense, white cataracts: a 10-month-old with congenital cataracts, a 9-year-old with a uveitic cataract, and a 17-year-old with a traumatic cataract. The aspirated material from the cataract surgeries contained insoluble material that could be isolated by centrifugation. Many particles within the insoluble fraction stained with Alizarin red, a dye that stains insoluble calcified material. The appearance of these human insoluble, Alizarin red-stained particles was similar to some of those detected in homogenates from cataractous mouse lenses. These results support the hypothesis that pathologic mineralization may have a mechanistic role in the formation of cataracts of different etiologies., Competing Interests: 5Conflict of Interest 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.

In the lens, cell homeostasis and transparency are supported by intercellular communication facilitated by the channels formed of connexin46 (Cx46) and connexin50 (Cx50). Mutations of these connexins are linked to inherited cataracts. We studied the levels and the variations in electrophoretic mobilities of the immunoreactive Cx46 and Cx50 bands between 1 and 21 days after birth in the lenses of wild-type mice and homozygous animals from two different mouse models of connexin-linked cataracts (Cx46fs380 and Cx50D47A). In Cx50D47A mice, the expression of the mutant Cx50 reduced the normal phosphorylation of the co-expressed wild-type Cx46. In both models, levels of the mutant connexin and the co-expressed wild-type connexin decayed more rapidly than in wild-type mice but with different time courses. In the Cx46fs380 mice, modeling suggested that Cx50 degradation could be explained by the mixing of mutant Cx46 with wild-type Cx50. However, in Cx50D47A mice, similar modeling suggested that mixing alone could not explain the decrease in Cx46 levels. These data highlight the complex influences between two connexin proteins expressed in the same cell, some of which occur through direct mixing, while others occur indirectly, as in Cx50D47A mice, where the expression of the mutant connexin causes endoplasmic reticulum stress and impaired differentiation.

Cataracts are lens opacities that are among the most common causes of blindness. It is commonly believed that cataracts develop through the accumulation of damage to lens proteins. However, recent evidence suggests that cataracts can result from calcium ion accumulation and the precipitation of calcium-containing salts. To test for the presence of precipitates and to identify their components, we studied the lenses of mice that develop cataracts due to mutations of connexin46 and connexin50. Micro-computed tomography showed the presence of radio-dense mineral in the mutant lenses, but not in wild-type lenses. Three-dimensional reconstructions of the scans showed that the distribution of the radio-dense mineral closely paralleled the location and morphology of the cataracts. The mutant lens homogenates also contained insoluble particles that stained with Alizarin red (a dye that stains Ca 2+ deposits). Using attenuated total internal reflection micro-Fourier transform infrared spectroscopy, we identified the mineral as calcium phosphate in the form of apatite. Taken together, these data support the novel paradigm that cataracts are formed through pathological mineralization within the lens., Competing Interests: 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., (Copyright © 2022 Minogue, Sommer, Williams, Bledsoe, Beyer and Berthoud.)

Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. Although most of these disease-linked variants cause loss of function because of misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Coexpression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Furthermore, when T39R was coexpressed with Cx46, it produced hemichannels with increased activity, particularly at negative potentials, which could potentially contribute to its pathogenicity in the lens. In contrast, coexpression of wild-type Cx50 with Cx46 was associated with a marked reduction in hemichannel activity, indicating that it may have a protective effect. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal (NT) domain while also neutralizing the voltage-sensing residue D3 as well as residue E42, which participates in loop gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cataracts of many different etiologies are associated with oxidation of lens components. The lens is protected by maintenance of a pool of reduced glutathione (GSH) and other antioxidants. Because gap junction channels made of the lens connexins, Cx46 and Cx50, are permeable to GSH, we tested whether mice expressing two different mutants, Cx46fs380 and Cx50D47A, cause cataracts by impairing lens glutathione metabolism and facilitating oxidative damage. Levels of GSH were not reduced in homogenates of whole mutant lenses. Oxidized glutathione (GSSG) and the GSSG/GSH ratio were increased in whole lenses of Cx50D47A, but not Cx46fs380 mice. The GSSG/GSH ratio was increased in the lens nucleus (but not cortex) of Cx46fs380 mice at 4.5 months of age, but it was not altered in younger animals. Carbonylated proteins were increased in Cx50D47A, but not Cx46fs380 lenses. Thus, both mouse lines have oxidizing lens environments, but oxidative modification is greater in Cx50D47A than in Cx46fs380 mice. The results suggest that GSH permeation through lens connexin channels is not a critical early event in cataract formation in these mice. Moreover, because oxidative damage was only detected in animals with significant cataracts, it cannot be an early event in their cataractogenesis.

Gap junction-mediated intercellular communication facilitates the circulation of ions, small molecules, and metabolites in the avascular eye lens. Mutants of the lens fiber cell gap junction proteins, connexin46 (Cx46) and connexin50 (Cx50), cause cataracts in people and in mice. Studies in mouse models have begun to elucidate the mechanisms by which these mutants lead to cataracts. The expression of the dominant mutants causes severe decreases in connexin levels, reducing the gap junctional communication between lens fiber cells and compromising the lens circulation. The impairment of the lens circulation results in several changes, including the accumulation of Ca 2+ in central lens regions, leading to the formation of precipitates that stain with Alizarin red. The cataract morphology and the distribution of Alizarin red-stained material are similar, suggesting that the cataracts result from biomineralization within the organ. In this review, we suggest that this may be a general process for the formation of cataracts of different etiologies.

Purpose: p62/Sequestosome 1 (p62) is a stress-induced protein that is involved in several different intracellular pathways, including regulation of aspects of protein degradation. p62 levels are elevated in several types of cataracts. We investigated whether levels of p62 and its phosphorylation were altered in the lenses of Cx50D47A mice, which express a mutant of connexin50 (Cx50) that leads to cataracts and impaired lens differentiation. To evaluate the importance of p62 in the lens defects caused by a connexin50 mutant, we also examined the effect of deleting p62 in homozygous Cx50D47A mice., Methods: Protein levels were determined with immunoblotting. Mouse lenses were examined with dark-field illumination microscopy. Intensities of the opacities and lens equatorial diameters were quantified using ImageJ. Nuclei and nuclear remnants were detected with fluorescence microscopy of lens sections stained with 4',6-diamino-2-phenylindole dihydrochloride (DAPI)., Results: Levels of total p62 were increased in the lenses of homozygous Cx50D47A mice compared to those of the wild-type animals. The ratio of p62 phosphorylated at threonine-269/serine-272 (T269/S272) to total p62 was significantly decreased, whereas the ratio of p62 phosphorylated at serine-349 (S349) to total p62 was significantly increased in lenses of homozygous Cx50D47A mice. However, deletion of p62 did not affect the sizes of the lenses or the severity of their cataracts in homozygous Cx50D47A mice. Deletion of p62 did not improve connexin50 or connexin46 levels. Moreover, deletion of p62 did not change the levels of crystallins, histone H3, the mitochondrial import receptor subunit TOM20 homolog, or the abundance of nuclei and nuclear fragments in the lenses of homozygous Cx50D47A mice. Homozygous deletion of p62 led to an 84% increase in the levels of ubiquilin 2, but did not significantly affect the levels of ubiquilin 1 or ubiquilin 4., Conclusions: Although homozygous Cx50D47A lenses have increased levels of p62, a specific reduction in p62 phosphorylation at T269/S272, and a specific increase in p62 phosphorylation at S349, this protein is not a critical determinant of the severity of the abnormalities of these lenses (reduced growth or differentiation and cataracts). The lens may utilize redundant or compensatory systems (such as changes in levels of ubiquilin 2) to compensate for the lack of p62 in homozygous Cx50D47A lenses., (Copyright © 2020 Molecular Vision.)

Purpose: CCAAT/enhancer-binding homologous protein (CHOP), a transcription factor that has been implicated in differentiation, apoptosis, and autophagy, is greatly elevated in lenses with cataracts due to mutations of several different lens proteins. To test the possible role of CHOP in the cataractous lens, we studied the effect of knocking out Chop in mice that were homozygous for the Cx50D47A mutation of the lens fiber gap junction protein connexin50 (Cx50)., Methods: Mouse lenses were examined by dark-field microscopy. Lens equatorial diameters and intensities of the opacities were quantified using ImageJ. Transcript levels were assessed by real-time quantitative PCR. Protein levels were determined by immunoblotting., Results: Homozygous Chop knockout lenses were transparent. Deletion of Chop in Cx50D47A mice did not improve lens transparency and had no effect on lens size. In Chop null-Cx50D47A lenses, the protein kinase R-like endoplasmic reticulum kinase (PERK)-dependent pathway was activated similarly to Cx50D47A lenses. In Cx50D47A mice, Chop deletion did not improve connexin levels or lens fiber cell differentiation, and it did not decrease the levels of Trib3 or Irs2 transcripts to wild-type values. However, homozygous Chop knockout significantly diminished the increased levels of Cebpb transcripts of Cx50D47A lenses., Conclusions: The results show that CHOP is not required for lens transparency. They also suggest that CHOP is not the critical etiological factor for the cataracts observed in homozygous Cx50D47A lenses, further supporting a major role for connexins in the disease., (Copyright © 2019 Molecular Vision.)

Purpose: Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation., Methods: Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+]i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images., Results: In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+]i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts., Conclusions: Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Mouse Cx50D47A and human Cx50D47N are non-functional connexin mutants that cause dominantly-inherited cataracts. In tissue culture expression experiments, they both exhibit impaired cellular trafficking and gap junction plaque formation. Lenses of mice expressing Cx50D47A have cataracts, reduced size, drastically decreased levels of connexin50, and less severely reduced levels of connexin46. The PERK-dependent pathway of the ER response to misfolded proteins is activated, and they have impaired differentiation with retained cellular organelles. Since treatments that enhance protein folding improve trafficking and plaque formation by Cx50D47N and other mutant connexins in vitro, and they are successful therapeutics for some other diseases caused by misfolded proteins, we tested the efficacy of the chemical chaperone, 4-phenylbutyrate (4-PBA) in cultured cells and mice expressing Cx50D47A. 4-PBA treatment increased the formation of Cx50D47A-containing plaques at appositional membranes of transiently transfected HeLa cells. Heterozygous Cx50D47A mice were treated with 4-PBA by addition to the drinking water and parenteral injection of pregnant mice (starting 10 days after pairing of males and females) and their pups. Lenses from 1-month-old mice were examined by darkfield illumination and immunofluorescence microscopy. Protein levels were determined by immunoblotting. Cataract size and density were not detectably different between the control and the 4-PBA-treated groups. Lens size was not increased following treatment. Levels of connexin46 and connexin50 were significantly increased in lenses of 4-PBA-treated mice compared with saline-treated animals. Immunofluorescence showed an increased abundance of connexin46 immunoreactivity and puncta. The ratio of phosphorylated to total EIF2α was not altered, and levels of organellar proteins were not significantly reduced, suggesting that the ER response to misfolded proteins and differentiation were not changed. Thus, treatment with 4-PBA improved critical pathological issues in these mice (low connexin and gap junction abundance), but the magnitude of this recovery (especially for Cx50) was inadequate to impact the reduced size or the opacification of Cx50D47A lenses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The lens is an avascular organ whose function and survival depend on an internal circulation system. Cx46fs380 mice model a human autosomal dominant cataract caused by a mutant lens connexin. In these mice, fiber cell connexin levels and gap junction coupling are severely decreased. The present studies were conducted to examine components of the lens circulation system that might be altered and contribute to the pathogenesis of cataracts. Lenses from wild-type mice and Cx46fs380 heterozygotes and homozygotes were studied at 2 months of age. Cx46fs380-expressing lens fiber cells were depolarized. Cx46fs380 lenses had increased intracellular hydrostatic pressure and concentrations of Na + and Ca 2+ . The activity of epithelial Na + -K + -ATPase was decreased in Cx46fs380 lenses. All of these changes were more severe in homozygous than in heterozygous Cx46fs380 lenses. Cx46fs380 cataracts were stained by Alizarin red, a dye used to detect insoluble Ca 2+ . These data suggest that the lens internal circulation was disrupted by expression of Cx46fs380, leading to several consequences including accumulation of Ca 2+ to levels so high that precipitates formed. Similar Ca 2+ -containing precipitates may contribute to cataract formation due to other genetic or acquired etiologies.

Purpose: Congenital cataracts occur in isolation in about 70% of cases or are associated with other abnormalities such as anterior segment dysgenesis and microphthalmia. We identified a three-generation family in the University of California San Francisco glaucoma clinic comprising three individuals with congenital cataracts and aphakic glaucoma, one of whom also had microphthalmia. The purpose of this study was to identify a possible causative mutation in this family and to investigate its pathogenesis., Methods: We performed exome sequencing and identified a putative mutation in gap junction protein α8 (GJA8). We used PCR and DNA sequencing of GJA8 in affected and unaffected members of the pedigree to test segregation of the variant with the phenotype. We tested cellular distribution and function of the variant protein by immunofluorescence and intercellular transfer of Neurobiotin in transiently transfected HeLa cells., Results: Exome sequencing revealed a variant in GJA8 (c.658A>G) encoding connexin50 (Cx50) that resulted in a missense change (p.N220D) in transmembrane domain 4. The variant was present in all three affected family members, but was also present in the proband's grandfather who was reported to be unaffected. The mutant protein localized to the plasma membrane and supported intercellular Neurobiotin transfer in HeLa cells., Conclusions: We identified a variant in transmembrane domain 4 of Cx50 in a family with autosomal dominant congenital cataracts. This variant has been previously identified in other cataract cohorts, but it is also present in unaffected individuals. Our study demonstrates that the mutant protein localized to the plasma membrane and formed functional intercellular channels. These data suggest that GJA8 c.658A>G is most likely a benign rare variant.

Purpose: Cx46fs380 mice model a human autosomal-dominant cataract caused by a mutant lens connexin46, Cx46. Lenses from Cx46fs380 mice develop cataracts that are first observed at ∼2 months in homozygotes and at ≥4 months in heterozygotes. The present studies were conducted to determine whether Cx46fs380 mouse lenses exhibited abnormalities before there are detectable cataracts., Methods: Lenses from wild-type and Cx46fs380 mice were studied at 1 to 3 months of age. Connexin levels were determined by immunoblotting. Gap junctional coupling was calculated from intracellular impedance studies of intact lenses. Optical quality and refractive properties were assessed by laser scanning and by photographing a 200-mesh electron microscopy grid through wild-type and Cx46fs380 mouse lenses., Results: Connexin46 and connexin50 levels were severely reduced in mutant lenses. Gap junctional coupling was decreased in differentiating and mature fibers from Cx46fs380 lenses; in homozygotes, the mature fibers had no detectable coupling. Homozygous lenses were slightly smaller and had reduced focal lengths. Heterozygous and homozygous lenses significantly distorted the electron microscopy grid pattern as compared with wild-type lenses., Conclusions: Before cataract appearance, Cx46fs380 lenses have decreased gap junctional conductance (at least in heterozygotes) and alterations in refractive properties (heterozygotes and homozygotes). The decreased focal distance of Cx46fs380 homozygous lenses is consistent with an increase in refractive index due to changes in cellular composition. These data suggest that Cx46fs380 lenses undergo a sequence of changes before the appearance of cataracts: low levels of connexins, decreased gap junction coupling, alterations in lens cell homeostasis, and changes in refractive index.

Mice expressing connexin50D47A (Cx50D47A) exhibit nuclear cataracts and impaired differentiation. Cx50D47A does not traffic properly, and homozygous mutant lenses show increased levels of the stress-responsive αB-crystallins. Therefore, we assessed whether expression of Cx50D47A led to endoplasmic reticulum (ER) stress in the lens in vivo Although pharmacologic induction of ER stress can be transduced by three different pathways, we found no evidence for activation of the IRE1α or ATF6 pathways in Cx50D47A-expressing lenses. In contrast, heterozygous and homozygous Cx50D47A lenses showed an increase in phosphorylated PERK immunoreactivity and in the ratio of phosphorylated to total EIF2α (2.4- and 3.3-fold, respectively) compared with wild type. Levels of ATF4 were similar in wild type and heterozygous lenses but elevated in homozygotes (391%). In both heterozygotes and homozygotes, levels of calreticulin protein were increased (184 and 262%, respectively), as was Chop mRNA (1.9- and 12.4-fold, respectively). CHOP protein was increased in homozygotes (384%). TUNEL staining was increased in Cx50D47A lenses, especially in homozygous mice. Levels of two factors that may be pro-survival, Irs2 and Trib3, were greatly increased in homozygous lenses. These results suggest that expression of Cx50D47A induces ER stress, triggering activation of the PERK-ATF4 pathway, which potentially contributes to the lens pathology and leads to increased expression of anti-apoptotic factors, allowing cell survival., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: To determine the basis of the autosomal dominant congenital cataracts in a three generation south Indian pedigree., Methods: The proband and several family members underwent a complete ophthalmic examination. The coding regions of eight candidate genes ( CRYAA, CRYBB2, CRYGC, CRYGD, GJA3, GJA8, AQP0, and PITX3 ) were amplified by PCR and directly sequenced. Wild type and mutant connexin50 (Cx50) were expressed by stable transfection of HeLa cells. Their cellular distributions and function were examined by immunofluorescence microscopy and by microinjection of gap junction permeant tracers, respectively., Results: Congenital cataracts (with some variations in phenotype) segregated as an autosomal dominant trait within a three generation pedigree. Three affected individuals (proband, sibling and mother) showed a sequence variation in the candidate gene GJA8 encoding Cx50: a c.1102G>C transversion encoding a substitution of glutamate for glutamine at position 368 (E368Q). This substitution was absent from an unaffected family member (paternal aunt) and 100 healthy controls of the same ethnicity. In transfected HeLa cells, both wild type Cx50 and E368Q localized to gap junction plaques, and supported similar levels of intercellular transfer of Neurobiotin., Conclusions: The E368Q mutant allele of GJA8 is associated with autosomal dominant congenital cataracts with phenotypic variability. E368Q forms gap junction plaques and functional channels in transfected HeLa cells.

An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48-72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+). Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.

Purpose: Although many connexin46 (Cx46) mutants have been linked to inherited human cataracts, there are no adequate animal models for their study. The current experiments were designed to characterize the consequences of expression of one such mutant, Cx46fs380, in the mouse lens., Methods: Mice expressing Cx46fs380 were generated by a knockin strategy. Levels and distribution of specific proteins were analyzed by immunoblotting and immunofluorescence., Results: Dark-field microscopy revealed that lenses of young heterozygous and homozygous Cx46fs380 mice did not have opacities, but they developed anterior nuclear cataracts that became more severe with age. Immunofluorescence and immunoblotting showed that Cx46 was severely reduced in both heterozygous and homozygous Cx46fs380 lenses at 1 month of age, whereas immunoreactive connexin50 (Cx50) was moderately decreased. The reduction in Cx50 became more severe in older lenses. The solubilities of crystallins from young wild-type and fs380 mice were similar, but older fs380 lenses exhibited abnormalities of abundance, solubility, and modification of some crystallins., Conclusions: Major decreases in connexin levels precede the development of cataracts. These mice represent a useful model for elucidation of the progression of lens abnormalities during cataractogenesis especially as caused by a mutant connexin., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Purpose: Substitutions of aspartate-47 (D47) of Connexin50 (Cx50) have been linked to autosomal dominant congenital cataracts in several human pedigrees. To elucidate the lens abnormalities caused by a substitution at this position, we studied No2 mice, which carry the Cx50D47A mutation and parallel the human pathology., Methods: Lenses from mice of different ages (neonatal to 4 months) were examined by dark-field and immunofluorescence microscopy. Protein levels were determined by immunoblotting using primary antibodies directed against connexins, other membrane proteins, crystallins, and proteins residing in different organelles., Results: Lenses of both heterozygous and homozygous Cx50D47A mice had cataracts and were smaller than those of wild-type littermates. Levels of Cx50 were severely reduced in mutant animals as compared with those in wild-type mice (<20% in heterozygotes and ≤3% in homozygotes). Levels of Cx46 and aquaporin0 were also decreased, but to a lesser extent. The immunostaining pattern of lens connexins was altered in mutant animals. The lenses of Cx50D47A mice showed persistence of nuclear remnants in deep regions of the lens and elevated levels of H3 histone and the mitochondrial protein, Tom20. γ-Crystallin levels were decreased in lenses of all mutant mice, and β-crystallins were reduced in homozygotes., Conclusions: These data suggest that mice expressing Cx50D47A develop cataracts due to a severe decrease in the abundance of functional connexin channels. They also implicate Cx50 in fiber cell differentiation, since mutant lenses showed impaired degradation of organelles and decreased levels of some crystallins.

The mechanisms by which mutant connexins lead to disease are diverse, including those of connexin50 (CX50) encoded by the GJA8 gene. We investigated the cellular and functional behavior of CX50fs, a mutant CX50 that has a frameshift after amino acid 255 and causes recessive congenital cataracts. Cellular levels of CX50fs were much lower than those of wild type CX50 in stably transfected HeLa cells. Whereas CX50 localized at distinct gap junction plaques and supported extensive intercellular transfer of Neurobiotin, CX50fs gap junctions were rare, and their support of Neurobiotin transfer was reduced by >90%. After inhibition of new protein synthesis with cycloheximide, CX50fs disappeared much more rapidly than CX50, suggesting increased degradation of the mutant. Treatment of cells with epoxomicin (a proteasomal inhibitor) led to a dramatic increase in CX50fs levels and in the abundance of gap junctions. Epoxomicin treatment also rescued intercellular transfer of Neurobiotin to levels similar to those in cells expressing the wild type protein. Treatment with eeyarestatin I (an inhibitor of p97-dependent protein degradation) resulted in many abundant slowly migrating CX50 and CX50fs bands consistent with polyubiquitination of the proteins. These results demonstrate that the CX50fs mutant is rapidly degraded by endoplasmic reticulum-associated degradation in mammalian cells. This accelerated degradation reduces the abundance of gap junctions and the extent of intercellular communication, potentially explaining the pathogenesis of cataracts linked to this mutant. The efficacy of epoxomicin in restoring function suggests that protease inhibition might have therapeutic value for this and other diseases caused by mutants with similar defects.

Autosomal dominant congenital cataracts have been associated with mutations of genes encoding several soluble and membrane proteins. By candidate gene screening, we identified a novel mutation in MIP (c.494 G > A) that segregates with a congenital lamellar cataract within a south Indian family and causes the replacement of a highly conserved glycine by aspartate (G165D) within aquaporin0 (AQP0). Unlike wild type AQP0, expression of AQP0-G165D in Xenopus oocytes did not facilitate swelling in hypotonic medium. In transfected HeLa cells, wild type AQP0 localized at the plasma membrane while AQP0-G165D was retained within the secretory pathway, and localized mainly within the endoplasmic reticulum. These results suggest that mutation of this conserved glycine residue leads to improper trafficking of AQP0-G165D and loss of water channel function. They emphasize the importance of AQP0 for maintenance of lens transparency and identify a critical residue that is conserved among aquaporins, but has not previously been associated with disease-associated replacement., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Various progenies of Eucalyptus grandis and E. amplifolia, and clones of Populus deltoides, were evaluated for plant removal of nitrogen (N) and phosphorus (P) for 26 months at a municipal waste spray field in north Florida. Tertiary treated wastewater containing 2.73 mg L(-1) nitrate N and 0.30 mg L(-1) total P was applied using sprinkler irrigation (93.8 m3 ha(-1) d(-1)) to fast growing trees utilized for bioenergy. Eucalyptus amplifolia and E. grandis survived and grew very poorly as the result of severe winter injury in two successive years and were not evaluated for nutrient removal. Survival and growth of P. deltoides demonstrated suitability for phytoremediation, and selected clones were evaluated for biomass and nutrient content. Removals of total N (TN) and total P (TP) were greatest for main stem (36% and 44%, respectively) and foliage (44% and 36%, respectively). Low biomass producing clones generally had higher nutrient concentrations, but high biomass producing clones removed more TN and TP. Approximately 789 kg ha(-1) TN and 103 kg ha(-1) TP were removed by the highest biomass producing P. deltoides clone, representing 215% of N and 615% of P inputs.

Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.

Oligomerization of connexins is a critical step in gap junction channel formation. Some members of the connexin family can oligomerize with other members and form functional heteromeric hemichannels [e.g. Cx43 (connexin 43) and Cx45], but others are incompatible (e.g. Cx43 and Cx26). To find connexin domains important for oligomerization, we constructed chimaeras between Cx43 and Cx26 and studied their ability to oligomerize with wild-type Cx43, Cx45 or Cx26. HeLa cells co-expressing Cx43, Cx45 or Cx26 and individual chimaeric constructs were analysed for interactions between the chimaeras and the wild-type connexins using cell biological (subcellular localization by immunofluorescence), functional (intercellular diffusion of microinjected Lucifer yellow) and biochemical (sedimentation velocity through sucrose gradients) assays. All of the chimaeras containing the third transmembrane domain of Cx43 interacted with wild-type Cx43 on the basis of co-localization, dominant-negative inhibition of intercellular communication, and altered sedimentation velocity. The same chimaeras also interacted with co-expressed Cx45. In contrast, immunofluorescence and intracellular diffusion of tracer suggested that other domains influenced oligomerization compatibility when chimaeras were co-expressed with Cx26. Taken together, these results suggest that amino acids in the third transmembrane domain are critical for oligomerization with Cx43 and Cx45. However, motifs in different domains may determine oligomerization compatibility in members of different connexin subfamilies.

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

The function of connexins, which form gap junctions, can be rapidly modulated by degradation, because they have half-lives of only a few hours. Autophagy is a degradation pathway that has been implicated in several diseases and can be induced by cellular stresses such as starvation. We investigated the involvement of autophagy in proteolysis of the wild-type connexins CX50 and CX43, and a cataract-associated connexin mutant, CX50P88S, which forms cytoplasmic accumulations. We observed that cytoplasmic connexins were partially (cup-shaped) or completely (ring-shaped) enclosed by structures containing the autophagy-related protein LC3. Intracellular connexins also colocalized with p62, a protein that might serve as a cargo receptor for autophagic degradation. Starvation induced a decrease in connexin levels that was blocked by treatment with chloroquine, a lysosomal protease inhibitor, or by knockdown of the autophagy-related protein Atg5. These results demonstrate that autophagy can regulate cellular levels of wild-type connexins and imply that the persistence of accumulations of CX50P88S results from insufficient degradation capacity of constitutive autophagy.

Purpose: To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts., Methods: The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp, Method: Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry., Results: Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions., Conclusions: Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.

Purpose: The aim of this study was the genetic, cellular, and physiological characterization of a connexin50 (CX50) variant identified in a child with congenital cataracts., Methods: Lens material from surgery was collected and used for cDNA production. Genomic DNA was prepared from blood obtained from the proband and her parents. PCR amplified DNA fragments were sequenced and characterized by restriction digestion. Connexin protein distribution was studied by immunofluorescence in transiently transfected HeLa cells. Formation of functional channels was assessed by two-microelectrode voltage-clamp in cRNA-injected Xenopus oocytes., Results: Ophthalmologic examination showed that the proband suffered from bilateral white, diffuse cataracts, but the parents were free of lens opacities. Direct sequencing of the PCR product produced from lens cDNA showed that the proband was heterozygous for a G>T transition at position 741 of the GJA8 gene, encoding the exchange of methionine for isoleucine at position 247 of CX50 (CX50I247M). The mutation was confirmed in the genomic DNA, but it was also present in the unaffected mother. When expressed in HeLa cells, both wild type CX50 and CX50I247M formed gap junction plaques. Both CX50 and CX50I247M induced gap junctional currents in pairs of Xenopus oocytes., Conclusions: Although the CX50I247M substitution has previously been suggested to cause cataracts, our genetic, cellular, and electrophysiological data suggest that this allele more likely represents a rare silent, polymorphic variant.

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly alpha-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the alpha-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.

The cytoplasmic N-termini of connexins have been implicated in protein trafficking, oligomerization and channel gating. To elucidate the role of the N-terminus in connexin37 (CX37), we studied mutant constructs containing partial deletions of its 23 N-terminal amino acids and a construct with a complete N-terminus in which residues 2-8 were replaced with alanines. All mutants containing nine or more N-terminal amino acids form gap junction plaques in transiently transfected HeLa cells, whereas most of the longer deletions do not. Although wild-type CX37 allowed intercellular transfer of microinjected neurobiotin in HeLa cells and formed conducting hemichannels in Xenopus oocytes, none of the mutant constructs tested show evidence of channel function. However, in coexpression experiments, N-terminal mutants that formed gap junction plaques potently inhibit hemichannel conductance of wild-type CX37 suggesting their co-oligomerization. We conclude that as much as half the length of the connexin N-terminus can be deleted without affecting formation of gap junction plaques, but an intact N-terminus is required for hemichannel gating and intercellular communication.

Purpose: To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23., Methods: Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double whole-cell patch-clamp recording., Results: HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer., Conclusions: These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.

Purpose: To screen for mutations of connexin50 (Cx50)/GJA8 in a panel of patients with inherited cataract and to determine the cellular and functional consequences of the identified mutation., Methods: All patients in the study underwent a full clinical examination and leucocyte DNA was extracted from venous blood. The GJA8 gene was sequenced directly. Connexin function and cellular trafficking were examined by expression in Xenopus oocytes and HeLa cells., Results: Screening of the GJA8 gene identified a 139 G to A transition that resulted in the replacement of aspartic acid by asparagine (D47N) in the coding region of Cx50. This change co-segregated with cataract among affected members of a family with autosomal dominant nuclear pulverulent cataracts. While pairs of Xenopus oocytes injected with wild type Cx50 RNA formed functional gap junction channels, pairs of oocytes injected with Cx50D47N showed no detectable intercellular conductance. Co-expression of Cx50D47N did not inhibit gap junctional conductance of wild type Cx50. In transiently transfected HeLa cells, wild type Cx50 localised to appositional membranes and within the perinuclear region, but Cx50D47N showed no immunostaining at appositional membranes with immunoreactivity confined to the cytoplasm. Incubation of HeLa cells transfected with Cx50D47N at 27 degrees C resulted in formation of gap junctional plaques., Conclusions: The pulverulent cataracts present in members of this family are associated with a novel GJA8 mutation, Cx50D47N, that acts as a loss-of-function mutation. The consequent decrease in lens intercellular communication and changes associated with intracellular retention of the mutant connexin may contribute to cataract formation.

To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken betaB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of alphaA-, alphaB-, beta- and gamma-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins.

An increasing number of diseases have been mapped to genes coding for ion channel proteins, including the gap junction proteins, connexins. Here, we report on the identification of an amino acid sequence underlying the behavior of a non-functional mutant connexin46 (CX46) associated with congenital cataracts. The mutant protein, CX46fs380, is 31 amino acids longer than CX46 and contains 87 aberrant amino acids in its C terminus. When expressed in mammalian cells, the mutant CX46 was not found at gap junctional plaques, but it showed extensive co-localization with markers for ERGIC and Golgi. The severe reductions in function and formation of gap junctional plaques were transferred to other connexins by creating chimeras containing the last third (or more) of the aberrant C terminus of the CX46 mutant. This sequence also impaired trafficking of a CD8 chimera. Site-directed mutagenesis of a diphenylalanine restored appositional membrane localization and function. These results suggest a novel mechanism in which a mutation causes disease by generating a motif that leads to retention within the synthetic/secretory pathway.

The gap junction protein connexin36 (CX36) has been well studied in the mature central nervous system, but there has been little information regarding its possible roles in embryonic development. We report here the isolation of the full-length chick CX36 coding sequence (predicted M(r) 35.1 kDa) and its strikingly restricted pattern of gene expression in the mesoderm of the chick embryo. In situ hybridization experiments demonstrated CX36 expression in somites by embryonic day 2. The transcripts first appeared dorsomedially within the somite and expanded ventrolaterally to form stripes in the middle of each somite. The CX36 stripes fell within somitic territories enriched in MYOD and FGF8 expression and impoverished in PAX3 transcripts, establishing that CX36 mRNA is expressed in the myotome. We compared the somitic expression pattern of CX36 with those of three other connexins, CX42, CX43, and CX45. At embryonic day 4, CX42 transcripts were localized to the myotome in a pattern resembling that of CX36. In contrast, CX43 was enriched in the dermomyotome, and CX45 was detected in both the myotome and the dermomyotome. Immunoblotting using Cx36 antibodies demonstrated bands of identical electrophoretic mobilities in trunk and retinal homogenates, and Cx36 immunostaining detected punctate immunoreactivity in the myotome. These results demonstrate that some connexins in the developing mesoderm are broadly expressed whereas others are highly localized, and suggest that CX36, CX42, and CX45 are involved in intercellular communication among developing muscle cells.

Gap junctional proteins, connexins, and gap junctional plaques are short-lived. Three pathways for their degradation have been proposed: (1) misfolded/abnormally oligomerized connexins are retrogradely translocated and degraded by the proteasome through endoplasmic reticulum-associated degradation; (2) connexins (as monomers or oligomers) may traffic directly from an early secretory compartment to the lysosome for degradation without reaching the plasma membrane; (3) connexins within gap junction plaques are degraded by the lysosome after endocytotic internalization. Degradation of gap junction plaques is proteasome-dependent in some cell types. Degradation may be regulated by ubiquitinylation, phosphorylation, or polypeptide domains that act as sorting signals.

Vitamin E (D,L-alpha-tocopherol) was administered to Paramecium tetraurelia in doses of 10, 100, 1000 and 10,000mg/l throughout its clonal lifespan. ANOVA revealed significant differences in clonal lifespan between groups, whether lifespan was measured in total fissions, or in days (P<0.05). When mean clonal lifespan was measured in fissions the greatest difference was between the 1000mg/l alpha-tocopherol treatment at 382 fissions, and the ethanol control at 255.5 fissions. The greatest difference in mean clonal lifespan in days survived was between the 10,000mg/l alpha-tocopherol treatment at 292.5 days and the ethanol control at 76 days. ANOVA also revealed significant differences (P<0.05) in the initial cell fission rates between groups. At the 1000 and 10,000mg/l concentrations of alpha-tocopherol, a decrease in cell fission rates was apparent early in the lifespan, but these rates began to increase gradually during the late clonal lifespan. Although no clonal toxicity effects were found in terms of decreasing life-expectancy, the 1000 and 10,000mg/l treatment groups exhibited higher background mortality rates throughout their respective lifespans than did the control groups, which could represent a cytotoxic effect.

A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when co-expressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.