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Xiaorong Xu, Lisa Ebihara; Characterization of a Mouse Cx50 Mutation Associated with the No2 Mouse Cataract. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1844-1850.
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purpose. Recently, a missense mutation in the mouse connexin 50 (Cx50) gene has been associated with the nuclear opacity
2 (No2) mouse cataract. This missense mutation (D47A) resulted
in an aspartate-to-alanine substitution at amino acid position 47 in
the first extracellular domain of Cx50. To better understand the role
of Cx50 in the pathogenesis of congenital cataract, the functional
consequences of the D47A mutation in the Xenopus oocyte
expression system were studied.
methods. D47A was constructed using polymerase chain reaction (PCR) mutagenesis. Xenopus oocytes were injected with in vitro transcribed
cRNA encoding wild-type mouse Cx50 (Cx50wt), wild-type rat Cx46
(Cx46wt), D47A, or combinations of wild-type and mutant connexins. The
oocytes were then devitellinized and paired. Gap junctional conductance
(G j ) was measured using a dual
two-microelectrode voltage-clamp technique.
results. Homotypic oocyte pairs expressing wild-type Cx50 or Cx46 were well
coupled. In contrast, oocytes injected with D47A cRNA did not form gap
junctional channels when paired homotypically. To test whether the D47A
mutation could interact with wild-type connexins in a dominant negative
manner, oocytes were injected with equal amounts of mutant and
wild-type connexin cRNA, mimicking the heterozygous condition.
Expression of D47A did not inhibit the development of junctional
conductance in paired oocytes induced by wild-type Cx50 or Cx46.
conclusions. These results indicate that the D47A mutation acts as a
loss-of-function mutation without strong dominant inhibition. In No2
mice, the mutation would be predicted to result in a reduction in
intercellular communication, leading to cataractogenesis. It may also
cause other qualitative changes such as a change in permeability for
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