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A. M. DeRosa, X. Gong, C.-H. Xia, T. W. White; The Cx50-S50P Mutation Dominantly Alters Wild-Type Cx46 Channel Gating. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4219.
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© ARVO (1962-2015); The Authors (2016-present)
Cx50 gene mutations 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 to examine the functional properties of gap junctions expressing a cataract causing Cx50 mutant protein, Cx50-S50P.
Murine Cx50 mutated at amino acid 50 (serine to proline) was subcloned in pCS2+, expressed in HeLa cells or transcribed in vitro and injected in paired Xenopus oocytes . Protein expression was confirmed, and quantified by immunoblotting and immunohistochemistry. The paired Xenopus oocyte system was used in conjunction with the dual whole-cell voltage clamp technique to analyze differences in macroscopic junctional conductance (Gj), channel kinetics, and voltage gating sensitivity.
These experiments revealed that Cx50-S50P subunits alone failed to induce electrical coupling. However, the mixed expression of Cx50-S50P and either wild-type Cx50 or Cx46 subunits, paired to create heteromeric gap junctions, formed functional intercellular channels with reduced conductance and altered voltage gating properties, when compared to homotypic wild-type channels. Additionally, immunofluorescent microscopy showed that Cx50-S50P subunits alone failed to localize to the plasma membrane, unlike Cx46 or Cx50 which concentrated at cell-cell appositions. In contrast, Cx50-S50P co-localized with wild-type Cx46 in cells expressing both proteins.
Taken together these data define, for the first time, the electrophysiological properties of functional gap junctions formed by the heteromeric combination of Cx50 or Cx46 and Cx50-S50P mutant proteins. Our results indicate that expression of Cx50-S50P alone fails to induce junctional coupling or efficiently form intercellular channels at the plasma membrane. Interestingly, the co-expression of Cx50-S50P with wild-type lens fiber connexins display altered gating properties a phenomenon that may contribute to the cause of cataract associated with this mutation.
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