Functional CX50 Hemichannels Are Responsible for a Congenital Cataract

Abstract

Purpose: : To determine the cellular and functional consequences of a CX50 mutation identified in a patient with autosomal dominant congenital total cataract.

Methods: : Ethical committee approval was granted and informed consent was obtained from all participants. The GJA8 gene was sequenced directly. Connexin-induced currents were recorded using two-electrode voltage clamp in Xenopus oocytes injected with wild type or mutant CX50 cRNAs. Protein expression and cellular localization of wild type or mutant CX50 were assessed by immunoblotting and immunofluorescence in transiently or stably transfected HeLa cells. Cell viability was assayed using a colorimetric assay (MTS). Cell cycle analysis was performed by flow cytometry.

Results: : Screening of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine with valine in the coding region of CX50 (CX50G46V). CX50G46V induced formation of gap junctional currents in pairs of Xenopus oocytes whose junction conductances were similar to those of wild-type CX50. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much greater than those in oocytes injected with similar amounts of wild type CX50 RNA. Transient transfection of HeLa cells with CX50G46V induced rounding up of cells with the immunoreactive protein localized throughout the cell. Because clones of HeLa cells stably expressing CX50G46V could not be obtained, stable transfectants were isolated in which the mutant CX50 was under the control of an inducible promoter. Both wild type CX50 and CX50G46V formed gap junctional plaques in induced HeLa cells as detected by immunofluorescence. Induction of CX50G46V led to changes in cell morphology, a decrease in cell number and reduced cell survival as quantified by MTS assay. Flow cytometric cell cycle analysis revealed an increase in the proportion of sub-G1 cells after induction of CX50G46V expression.

Conclusions: : These results demonstrate that CX50G46V, unlike other cataract-associated CX50 mutants, traffics properly to the plasma membrane and can form functional connexin hemichannels and gap junction channels; however, its expression leads to apoptosis. They suggest a novel mechanism by which connexin mutants lead to cataracts: gain of hemichannel function.

Keywords: cataract • gap junctions/coupling • apoptosis/cell death