A crystallin mutant cataract with mineral deposits.

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 (Crygcdup). 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 Crygcdup 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.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-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.

Cataract-linked serine mutations in the gap junction protein connexin50 expose a sorting signal that promotes its lysosomal degradation.

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.

Molecular mechanisms underlying enhanced hemichannel function of a cataract-associated Cx50 mutant.

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.

p62/Sequestosome 1 levels increase and phosphorylation is altered in Cx50D47A lenses, but deletion of p62/sequestosome 1 does not improve transparency.

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.

Connexin Mutants Compromise the Lens Circulation and Cause Cataracts through Biomineralization.

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 Ca2+ 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.

CHOP is dispensable for lens transparency in wild-type and connexin50 mutant mice.

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.

Disruption of the lens circulation causes calcium accumulation and precipitates in connexin mutant mice.

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 Ca2+. 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 Ca2+. These data suggest that the lens internal circulation was disrupted by expression of Cx46fs380, leading to several consequences including accumulation of Ca2+ to levels so high that precipitates formed. Similar Ca2+-containing precipitates may contribute to cataract formation due to other genetic or acquired etiologies.

Gap junction gene and protein families: Connexins, innexins, and pannexins.

Gap junction channels facilitate the intercellular exchange of ions and small molecules. While this process is critical to all multicellular organisms, the proteins that form gap junction channels are not conserved. Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. Interestingly, vertebrates and lower chordates contain innexin homologs, the pannexins, which also form channels, but rarely (if ever) make intercellular channels. While the connexin and the innexin/pannexin polypeptides do not share significant sequence similarity, all three of these protein families share a similar membrane topology and some similarities in quaternary structure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Chemical chaperone treatment improves levels and distributions of connexins in Cx50D47A mouse lenses.

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.

The Cataract-linked Mutant Connexin50D47A Causes Endoplasmic Reticulum Stress in Mouse Lenses.

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.

Focus on lens connexins.

The lens is an avascular organ composed of an anterior epithelial cell layer and fiber cells that form the bulk of the organ. The lens expresses connexin43 (Cx43), connexin46 (Cx46) and connexin50 (Cx50). Epithelial Cx50 has critical roles in cell proliferation and differentiation, likely involving growth factor-dependent signaling pathways. Both Cx46 and Cx50 are crucial for lens transparency; mutations in their genes have been linked to congenital and age-related cataracts. Congenital cataract-associated connexin mutants can affect protein trafficking, stability and/or function, and the functional effects may differ between gap junction channels and hemichannels. Dominantly inherited cataracts may result from effects of the connexin mutant on its wild type isotype, the other co-expressed wild type connexin and/or its interaction with other cellular components.

Connexin23 deletion does not affect lens transparency.

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.

The connexin46 mutant, Cx46T19M, causes loss of gap junction function and alters hemi-channel gating.

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.

A connexin50 mutant, CX50fs, that causes cataracts is unstable, but is rescued by a proteasomal inhibitor.

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.

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Autophagy: a pathway that contributes to connexin degradation.

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.

An MIP/AQP0 mutation with impaired trafficking and function underlies an autosomal dominant congenital lamellar cataract.

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.

Pediatric cataracts of different etiologies contain insoluble, calcified particles.

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.

Cytoplasmic amino acids within the membrane interface region influence connexin oligomerization.

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.