Molecular Mechanism Underlying a Lamellar Pulverulent Cataract Associated With a Novel Connexin50 Mutation

Abstract

Abstract: : Purpose: To determine the cellular and functional consequences of a Cx50 mutation identified in a family with autosomal dominant lamellar pulverulent cataract. Methods: Wild type hCx50 and hCx50P88Q were expressed in Xenopus oocytes and gap junction currents were recorded using the double whole–cell voltage–clamp technique. Stable clones of HeLa cells were obtained after transfection with hCx50 or hCx50P88Q subcloned into pcDNA3.1–Hygro. The cellular distributions of hCx50 and hCx50P88Q were determined by immunofluorescence microscopy. Results: hCx50 formed functional channels between Xenopus oocytes with a gap junction conductance of 4.02 + 0.368 µS. In contrast, pairs of oocytes injected with the mutant hCx50P88Q showed no detectable gap junction conductance (0.127 + 0.030 µS). When co–expressed with wild type hCx50, hCx50P88Q significantly inhibited gap junctional activity of wild type hCx50, reducing the conductance by 66–75% when compared with oocyte pairs expressing wild type hCx50 alone. Using anti–Cx50 antibodies, HeLa cells transfected with hCx50 showed immunopositive staining at appositional membranes and in the perinuclear region as expected. In contrast, cells transfected with hCx50P88Q showed little or no anti–Cx50 immunostaining at appositional membranes, but showed substantial immunoreactivity in the cytoplasm. Partial colocalisation of anti–Cx50 immunoreactivity with markers for ER or Golgi compartments was observed in cells transfected with hCx50P88Q. Incubation of hCx50P88Q–transfected cells at 30°C increased immunoreactivity at appositional membranes. Conclusions: hCx50P88Q does not form functional gap junction channels in Xenopus oocytes or gap junction plaques when transfected into HeLa cells. The increased immunoreactivity at appositional membranes observed after incubation at 30°C suggests that hCx50P88Q has a folding/trafficking defect. These results suggest that the improper trafficking/folding of hCx50P88Q leads to a decrease in intercellular communication and cataract formation.

Keywords: gap junctions/coupling • cataract