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T.W. White, A.M. DeRosa, C. Sellitto, D. Cheung, C.–H. Xia, X. Gong; Rescue of a Semi–Dominant Connexin50 Cataract (Lop10) by Knockin of Wild–Type Connexin46 . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5416.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in Cx46 and Cx50 cause cataracts in humans and mice and the mechanisms whereby mutated connexins lead to cataract are poorly understood. We previously showed that heterozygous replacement of wild–type Cx50 with wild–type Cx46 by genetic knockin [Cx50(+/46)] resulted in dominant cataracts despite providing robust coupling (JCB 161: 969; IOVS 45:3629). Lop10 is a dominant mouse cataract caused by a point mutation in the Cx50 gene (G22R, Hum Mol Genet 11:507). To further evaluate the role of connexins in cataractogenesis, we have examined the ability of knockin Cx46 to rescue the severe cataract caused by the Cx50–G22R mutation.
Three compound mutant lines, [Cx50(G22R/–) Cx46(–/–)], [Cx50(46/–) Cx46(–/–)] and [Cx50(G22R/46) Cx46(–/–)] were generated from breeding lop10, knockin Cx46, and Cx46/Cx50 double knockout mice. Lenses were examined by light microscopy. Connexin distribution was analyzed by immunohistochemistry. G22R and Cx46 were expressed in paired Xenopus oocytes and dual whole–cell voltage clamp was used to measure junctional conductance and gating.
Lenses expressing one G22R allele [Cx50(G22R/–) Cx46(–/–)] had severely disorganized fibers and posterior rupture, while lenses with one knockin Cx46 allele [Cx50(46/–) Cx46(–/–)] remained clear. Heterozygous mixing of G22R with knockin Cx46 [Cx50(G22R/46) Cx46(–/–)] rescued the lens rupture and cataract and targeted G22R to gap junction plaques. In paired Xenopus oocytes, Cx50–G22R alone failed to induce electrical coupling. However, mixed expression of Cx50–G22R and wild–type Cx46 produced functional channels with reduced conductance and altered voltage sensitivity when compared to channels formed by Cx46 alone.
Forced mixing of knockin Cx46 and Cx50–G22R on the Cx50 gene locus rescued the lens rupture phenotype by stabilizing Cx50–G22R and facilitating its targeting to lens fiber gap junctions. This is in sharp contrast to the dominant cataracts that previously resulted from forced mixing of knockin Cx46 and wild–type Cx50. Co–expression of Cx46 and G22R produced functional channels with novel physiological properties suggesting that formation of heteromeric gap junction channels containing knockin Cx46 and Cx50–G22R contributed to the rescue of the dominant lop10 phenotype. These results demonstrate that the functional nature of gap junction coupling profoundly influences cataractogenesis.
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