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Originally published In Press as doi:10.1167/iovs.09-3759 on August 13, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:5837-5845.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.09-3759
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A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death

Peter J. Minogue,1 Jun-Jie Tong,2 Anita Arora,3,4 Isabelle Russell-Eggitt,5 David M. Hunt,3 Anthony T. Moore,3,4,5 Lisa Ebihara,2 Eric C. Beyer,1 and Viviana M. Berthoud1

From the 1Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois; the 2Department of Physiology and Biophysics, Rosalind Franklin School of Medicine and Science, North Chicago, Illinois; 3UCL Institute of Ophthalmology, London, United Kingdom; the 4Moorfields Eye Hospital, London, United Kingdom; and the 5Great Ormond Street Hospital for Children, London, United Kingdom.

Corresponding author: Viviana M. Berthoud, Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, 5841 S. Maryland Avenue, MC 4060, Chicago, IL 60637; vberthou{at}peds.bsd.uchicago.edu.

Purpose. To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts.

Methods. The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp method. Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry.

Results. Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions.

Conclusions. Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.






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