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Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Published on: July 16, 2013
A mutant connexin50 with enhanced hemichannel function leads to cell death
Peter J Minogue1, Jun-Jie Tong, Anita Arora
1Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois 60637, USA.
Investigative Ophthalmology & Visual Science
|August 18, 2009
Summary
A novel connexin50 (CX50) mutant, CX50G46V, identified in a child with congenital cataracts, causes cell death through enhanced hemichannel function. This finding suggests a new mechanism for connexin-related cataracts.
Area of Science:
- Ophthalmology
- Cell Biology
- Genetics
Background:
- Congenital cataracts can result from genetic mutations affecting lens proteins.
- Connexins, particularly connexin50 (CX50), are crucial for lens transparency and function.
- Mutations in the GJA8 gene encoding CX50 are linked to various forms of cataracts.
Purpose of the Study:
- To investigate the functional consequences of a novel CX50 mutant (CX50G46V) found in a patient with congenital total cataracts.
- To elucidate the cellular mechanisms underlying the pathogenicity of this CX50 mutation.
Main Methods:
- Direct sequencing of the GJA8 gene to identify mutations.
- Two-microelectrode voltage-clamp electrophysiology to assess channel function in Xenopus oocytes.
- Immunoblot analysis and immunofluorescence to evaluate protein expression and localization.
- Flow cytometry to quantify apoptosis in HeLa cells.
Main Results:
- A GJA8 gene mutation identified a CX50G46V mutant.
- CX50G46V formed functional gap junction and hemichannel currents in Xenopus oocytes, with significantly enhanced hemichannel activity upon calcium removal.
- Expression of CX50G46V in HeLa cells led to cell death, characterized by increased apoptosis, which was mitigated by high extracellular calcium.
- Both wild-type CX50 and CX50G46V trafficked to the plasma membrane and formed gap junction plaques.
Conclusions:
- The CX50G46V mutant properly traffics to the plasma membrane and forms functional channels.
- Unlike previously studied CX50 mutants, CX50G46V exhibits potent cytotoxicity, even at low expression levels, primarily due to enhanced hemichannel function.
- This suggests a novel mechanism where connexin-mediated cytotoxicity, driven by aberrant hemichannel activity, contributes to the development of congenital cataracts.
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