Peter J. Minogue

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.

Loss of function and impaired degradation of a cataract-associated mutant connexin50

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.

A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract

Purpose: To identify the gene responsible for autosomal dominant lamellar pulverulent cataract in a four-generation British family and characterise the functional and cellular consequences of the mutation. Methods: Linkage analysis was used to identify the disease locus. The GJA8 gene was sequenced directly. Functional behaviour and cellular trafficking of connexins were examined by expression in Xenopus oocytes and HeLa cells. Results: A 262C>A transition that resulted in the replacement of proline by glutamine (P88Q) in the coding region of connexin50 (Cx50) was identified. hCx50P88Q did not induce intercellular conductance and significantly inhibited gap junctional activity of co-expressed wild type hCx50 RNA in paired Xenopus oocytes. In transfected cells, immunoreactive hCx50P88Q was confined to the cytoplasm but showed a temperature sensitive localisation at gap junctional plaques. Conclusions: The pulverulent cataract described in this family is associated with a novel GJA8 mutation and has a different clinical phenotype from previously described GJA8 mutants. The cataract likely results from lack of gap junction function. The lack of function was associated with improper targeting to the plasma membrane, most probably due to protein misfolding.

A mutant connexin50 with enhanced hemichannel function leads to cell death.

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.

A novel connexin50 mutation associated with congenital nuclear pulverulent cataracts

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°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.

An Aberrant Sequence in a Connexin46 Mutant Underlies Congenital Cataracts

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.

An intact connexin N-terminus is required for function but not gap junction formation.

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.

Cataracts are caused by alterations of a critical N-terminal positive charge in connexin50.

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.

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

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.

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.