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      Targeted Ablation of Connexin50 in Mice Results in Microphthalmia and Zonular Pulverulent Cataracts

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          Abstract

          In the ocular lens, gap junctional communication is a key component of homeostatic mechanisms preventing cataract formation. Gap junctions in rodent lens fibers contain two known intercellular channel-forming proteins, connexin50 (Cx50) and Cx46. Since targeted ablation of Cx46 has been shown to cause senile-type nuclear opacities, it appears that Cx50 alone cannot meet homeostatic requirements. To determine if lens pathology arises from a reduction in levels of communication or the loss of a connexin-specific function, we have generated mice with a targeted deletion of the Cx50 gene. Cx50-null mice exhibited microphthalmia and nuclear cataracts. At postnatal day 14 (P14), Cx50-knockout eyes weighed 32% less than controls, whereas lens mass was reduced by 46%. Cx50-knockout lenses also developed zonular pulverulent cataracts, and lens abnormalities were detected by P7. Deletion of Cx50 did not alter the amounts or distributions of Cx46 or Cx43, a component of lens epithelial junctions. In addition, intercellular passage of tracers revealed the persistence of communication between all cell types in the Cx50-knockout lens. These results demonstrate that Cx50 is required not only for maintenance of lens transparency but also for normal eye growth. Furthermore, these data indicate that unique functional properties of both Cx46 and Cx50 are required for proper lens development.

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          The gap junction communication channel.

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            Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes

            Gap junctions are composed of a family of structural proteins called connexins, which oligomerize into intercellular channels and function to exchange low molecular weight metabolites and ions between adjacent cells. We have cloned a new member of the connexin family from lens cDNA, with a predicted molecular mass of 46 kD, called rat connexin46 (Cx46). Since a full-length cDNA corresponding to the 2.8-kb mRNA was not obtained, the stop codon and surrounding sequences were confirmed from rat genomic DNA. The RNA coding for this protein is abundant in lens fibers and detectable in both myocardium and kidney. Western analysis of both rat and bovine lens membrane proteins, using the anti- MP70 monoclonal antibody 6-4-B2-C6 and three anti-peptide antibodies against Cx46 demonstrates that Cx46 and MP70 are different proteins. Immunocytochemistry demonstrates that both proteins are localized in the same lens fiber junctional maculae. Synthesis of Cx46 in either reticulocyte lysate or Xenopus oocytes yields a 46-kD polypeptide; all anti-Cx46 antisera recognize a protein in rat lens membranes 5-10 kD larger, suggesting substantive lenticular posttranslational processing of the native translation product. Oocytes that have synthesized Cx46 depolarize and lyse within 24 h, a phenomenon never observed after expression of rat connexins 32 or 43 (Cx32 and Cx43). Lysis is prevented by osmotically buffering the oocytes with 5% Ficoll. Ficoll- buffered oocytes expressing Cx46 are permeable to Lucifer Yellow but not FITC-labeled BSA, indicating the presence of selective membrane permeabilities. Cx43-expressing oocytes are impermeable to Lucifer Yellow. Voltage-gated whole cell currents are measured in oocytes injected with dilute concentrations of Cx46 but not Cx43 mRNA. These currents are activated at potentials positive to -10 mV. Unlike other connexins expressed in Xenopus oocytes, these results suggest that unprocessed Cx46 induces nonselective channels in the oolemma that are voltage dependent and opened by large depolarizations.
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              Connections with connexins: the molecular basis of direct intercellular signaling.

              Adjacent cells share ions, second messengers and small metabolites through intercellular channels which are present in gap junctions. This type of intercellular communication permits coordinated cellular activity, a critical feature for organ homeostasis during development and adult life of multicellular organisms. Intercellular channels are structurally more complex than other ion channels, because a complete cell-to-cell channel spans two plasma membranes and results from the association of two half channels, or connexons, contributed separately by each of the two participating cells. Each connexon, in turn, is a multimeric assembly of protein subunits. The structural proteins comprising these channels, collectively called connexins, are members of a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure/function studies have begun to provide a molecular understanding of the significance of connexin diversity and demonstrated the unique regulation of connexins by tyrosine kinases and oncogenes. Finally, mutations in two connexin genes have been linked to human diseases. The development of more specific approaches (dominant negative mutants, knockouts, transgenes) to study the functional role of connexins in organ homeostasis is providing a new perception about the significance of connexin diversity and the regulation of intercellular communication.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                2 November 1998
                : 143
                : 3
                : 815-825
                Affiliations
                Department of Cell Biology and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
                Author notes

                Address correspondence to David L. Paul, Department of Neurobiology, Harvard Medical School, Boston, MA 02115. Tel.: (617) 432-1203. Fax: (617) 432-2955. E-mail: dpaul@ 123456hms.harvard.edu

                Article
                10.1083/jcb.143.3.815
                2148149
                9813099
                28eeca82-e8d4-4f08-823c-2215a9bf81b4
                Copyright @ 1998
                History
                : 27 April 1998
                : 10 September 1998
                Categories
                Regular Articles

                Cell biology
                lens,gap junction,connexin,microphthalmia,cataract
                Cell biology
                lens, gap junction, connexin, microphthalmia, cataract

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