Search

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.

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.

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.

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.

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.