RESUMO
The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8(R205G) point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8(R205G/R205G) mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8(R205G) mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8(R205G) mutation occurs at the same conserved residue as the human GJA8(R198W) mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.
Assuntos
Catarata/genética , Conexinas/genética , Proteínas do Olho/genética , Microftalmia/genética , Mutação de Sentido Incorreto , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Catarata/metabolismo , Catarata/patologia , Forma Celular , Células Cultivadas , Conexinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Proteínas do Olho/metabolismo , Feminino , Genes Dominantes , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Humanos , Ativação do Canal Iônico , Cristalino/patologia , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Microftalmia/metabolismo , Microftalmia/patologia , Análise de Sequência de DNA , Vacúolos/metabolismo , Xenopus laevisRESUMO
We have identified and characterized a zebrafish connexin, Cx30.3. Sequence similarity analyses suggested that Cx30.3 was orthologous to both mammalian Cx26 and Cx30, known to play important roles in the skin and inner ear of mammals. Analysis of mRNA expression showed that Cx30.3 was present in early embryos, and was highly abundant in skin, but also detected in other tissues including fins, inner ear, heart, and the retina. Injection of Cx30.3 cRNA into Xenopus oocytes elicited robust intercellular coupling with voltage gating sensitivity similar to mammalian Cx26 and Cx30. The similarities in functional properties and expression patterns suggest that Cx30.3, like mammalian Cx26 and Cx30, may play a significant role in skin development, hearing, and balance in zebrafish. Thus, zebrafish could potentially serve as an excellent model to study disorders of the skin and deafness that result from human connexin mutations.
Assuntos
Conexinas/metabolismo , Pele/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Conexina 26 , Conexinas/química , Conexinas/classificação , Conexinas/genética , Orelha Interna/embriologia , Orelha Interna/metabolismo , Embrião não Mamífero/metabolismo , Éxons/genética , Humanos , Hibridização In Situ , Íntrons/genética , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Pele/embriologia , Sítio de Iniciação de Transcrição , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/genéticaRESUMO
Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling >/=90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.
Assuntos
Catarata/genética , Comunicação Celular/fisiologia , Conexina 43/metabolismo , Conexinas/genética , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Cristalino/citologia , Cristalino/metabolismo , Sequência de Aminoácidos , Animais , Catarata/patologia , Conexina 43/genética , Conexinas/metabolismo , Células Epiteliais/citologia , Proteínas do Olho/metabolismo , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Cristalino/patologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Conformação Proteica , Xenopus laevisRESUMO
Mutations in the GJB2 gene-encoding connexin 26 (Cx26) have been linked to skin disorders and genetic deafness. However, the severity and type of the skin disorders caused by Cx26 mutations are heterogeneous. Here we explored the effect of Cx26 KID syndrome-associated mutations, G12R, S17F, and D50N on channel function. The Cx26 N14K mutation was also examined that is associated with deafness but has a skin disorder distinct from the KID syndrome mutations. The proteins were all expressed in Xenopus oocytes with levels equal to wild-type Cx26. The G12R, N14K, and D50N mutations resulted in larger hemichannel currents than the wild-type-expressing cells, but the S17F mutation resulted in a complete loss of hemichannel activity. Elevated hemichannel activity correlated with an increased cell death. This result could be reversed through the elevation of calcium (Ca2+) in the extracellular media. Functional gap junctions were only produced by paired N14K cells, which had a similar conductance level to wild type, even though they exhibited a complete loss of voltage sensitivity. This set of data confirms that aberrant hemichannel activity is a common feature of Cx26 mutations associated with KID syndrome, and this may contribute to a loss of cell viability and tissue integrity.
Assuntos
Apoptose , Conexinas/genética , Surdez/genética , Junções Comunicantes/fisiologia , Ictiose/genética , Ceratite/genética , Mutação , Animais , Cálcio/fisiologia , Conexina 26 , Conexinas/fisiologia , Surdez/etiologia , Humanos , Ictiose/etiologia , Ceratite/etiologia , Oócitos/metabolismo , XenopusRESUMO
Mutations within connexin50 (Cx50) have been linked to various cataract phenotypes. To determine the mechanism behind cataract formation we used the paired Xenopus oocyte system in conjunction with transfected HeLa cells and genetically engineered mouse models to examine the functional characteristics of gap junctions in which a cataract-causing mutant of Cx50 (hereafter referred to as Cx50-S50P) is expressed. Channels comprising Cx50-S50P subunits alone failed to induce electrical coupling. However, the mixed expression of Cx50-S50P and wild-type subunits of either Cx50 or Cx46 - to create heteromeric gap junctions - resulted in functional intercellular channels with altered voltage-gating properties compared with homotypic wild-type channels. Additionally, immunofluorescence microscopy showed that channels of Cx50-S50P subunits alone failed to localize to the plasma membrane - unlike channels composed of Cx46 subunits, which concentrated at cell-cell appositions. Cx50-S50P colocalized with wild-type Cx46 in both transfected HeLa cells in vitro and mouse lens sections in vivo. Taken together, these data define the electrophysiological properties and intracellular targeting of gap junctions formed by the heteromeric combination of Cx50 or Cx46 and Cx50-S50P mutant proteins. Additionally, mixed channels displayed significantly altered gating properties, a phenomenon that may contribute to the cataract that is associated with this mutation.
Assuntos
Catarata/genética , Conexinas/genética , Proteínas do Olho/genética , Ativação do Canal Iônico/fisiologia , Cristalino/metabolismo , Mutação , Animais , Catarata/patologia , Conexinas/metabolismo , Conexinas/fisiologia , Eletrofisiologia , Proteínas do Olho/fisiologia , Feminino , Junções Comunicantes/genética , Junções Comunicantes/patologia , Engenharia Genética/métodos , Células HeLa , Heterozigoto , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microinjeções , Modelos Biológicos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Transfecção , XenopusRESUMO
Mutations in the zebrafish connexin43 (cx43) gene cause the short fin phenotype, indicating that direct cell-cell communication contributes to bone length. Three independently generated cx43 alleles exhibit short segments of variable sizes, suggesting that gap junctional intercellular communication may regulate bone growth. Dye coupling assays showed that all alleles are capable of forming gap junction channels. However, ionic coupling assays revealed allele-specific differences in coupling efficiency and gating. For instance, oocyte pairs expressing the weakest allele exhibited much higher levels of coupling than either of the strong alleles. Therefore, measurable differences in Cx43 function may be correlated with the severity of defects in bone length.
Assuntos
Comunicação Celular/genética , Conexina 43/genética , Junções Comunicantes/genética , Deformidades Congênitas dos Membros/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Ossos da Extremidade Inferior/anormalidades , Células HeLa , Humanos , Mutação de Sentido Incorreto/fisiologia , Técnicas de Patch-Clamp , Fenótipo , TransfecçãoRESUMO
Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca(2+) levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed.
Assuntos
Conexinas/metabolismo , Surdez/metabolismo , Junções Comunicantes/metabolismo , Ictiose/metabolismo , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Ceratite/metabolismo , Mutação , Animais , Cálcio/metabolismo , Morte Celular , Conexina 26 , Conexinas/genética , Surdez/genética , Surdez/patologia , Surdez/fisiopatologia , Líquido Extracelular/metabolismo , Feminino , Junções Comunicantes/genética , Predisposição Genética para Doença , Genótipo , Homeostase , Humanos , Ictiose/genética , Ictiose/patologia , Ictiose/fisiopatologia , Ativação do Canal Iônico/genética , Canais Iônicos/genética , Ceratite/genética , Ceratite/patologia , Ceratite/fisiopatologia , Potenciais da Membrana , Microinjeções , Oócitos/metabolismo , Oócitos/patologia , Fenótipo , Síndrome , Xenopus laevisRESUMO
PURPOSE: Lens connexins undergo proteolytic cleavage of their C termini during fiber maturation. Although the functional significance of this is unknown, cleavage has been correlated with changes in channel-gating properties. This study evaluates the functional consequences of this endogenous truncation by characterizing the properties of a C-terminal truncated Cx50 protein. METHODS: Murine and human Cx50 were truncated at amino acids 290 and 294, respectively, before expression in paired Xenopus oocytes or mammalian cells. Protein expression was evaluated by immunocytochemistry. Dual whole-cell voltage clamp techniques were used to analyze macroscopic and single-channel conductance, voltage-gating properties, and kinetics; pH gating sensitivity was measured by superfusion with 100% CO2-saturated media. RESULTS: Cx50tr290 channels exhibited an 86% to 89% reduction in mean macroscopic conductance compared with full-length Cx50. Heterotypic channels formed functional gap junctions, displayed an intermediate level of coupling, and exhibited unaltered voltage-gating properties. C-terminal truncation did not alter single-channel gating characteristics or unitary conductance. Interestingly, truncated and full-length Cx50 channel conductances were reversibly blocked by cytoplasmic acidification. CONCLUSIONS: C-terminal truncation of Cx50 did not inhibit the formation of homotypic or heterotypic channels. However, a significant decrease in conductance was observed for truncated channels, a phenomenon independent of alterations in voltage-gating sensitivity, kinetics, or chemical gating. These results provide a plausible explanation for the 50% decrease in junctional coupling observed during lens fiber maturation.
Assuntos
Conexinas/fisiologia , Proteínas do Olho/fisiologia , Junções Comunicantes/fisiologia , Expressão Gênica/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Animais , Western Blotting , Clonagem Molecular , Eletrofisiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Oócitos/fisiologia , Técnicas de Patch-Clamp , Transfecção , Xenopus laevisRESUMO
A G22R point mutation in alpha8 connexin (Cx50) has been previously shown to cause a severe cataract by interacting with endogenous wild-type alpha3 connexin (Cx46) in mouse lenses. Here, we tested whether a knocked-in alpha3 connexin expressed on the locus of the endogenous alpha8 connexin could modulate the severe cataract caused by the alpha8-G22R mutation. We found that the alpha3(-/-) alpha8(G22R/-) mice developed severe cataracts with disrupted inner fibers and posterior rupture while the alpha3(-/-) alpha8(G22R/KIalpha3) lens contained relatively normal inner fibers without lens posterior rupture. The alpha8-G22R mutant proteins produced typical punctate staining of gap junctions between fiber cells of alpha3(-/-) alpha8(G22R/KIalpha3) lenses, but not in those of alpha3(-/-) alpha8(G22R/-) lenses. Thus, we hypothesize that the knocked-in alpha3 connexin subunits interact with the alpha8-G22R connexin subunits to form functional gap junction channels and rescue the lens phenotype. Using an electrical coupling assay consisting of paired Xenopus oocytes, we demonstrated that only co-expression of mutant alpha8-G22R and wild-type alpha3 connexin subunits forms functional gap junction channels with reduced conductance and altered voltage sensitivity compared with the channels formed by alpha3 connexin subunits alone. Thus, knocked-in alpha3 connexin and mutant alpha8-G22R connexin probably form heteromeric gap junction channels that influence lens homeostasis and lens transparency.