Abstract
Purpose:
To elucidate the cellular and functional defects of CX50fs, a human Connexin50 (CX50) mutant that contains a frame shift after amino acid 255 and is linked to a recessive congenital cataract.
Methods:
DNA encoding CX50fs was obtained by PCR, subcloned into a mammalian expression vector, and expressed by stable transfection of HeLa cells. Levels and distribution of connexins were assessed by immunoblotting and immunofluorescence. Gap junction-mediated intercellular communication was evaluated by transfer of Neurobiotin after microinjection into one cell of a cluster.
Results:
Cellular levels of CX50fs were lower than those of wild type CX50. While CX50 localized at distinct gap junctional plaques, CX50fs only formed rare and barely detectable gap junctional plaques. In cells expressing CX50, we observed on average intercellular transfer of Neurobiotin to >100 neighboring cells, compared to the limited transfer supported by CX50fs to ~10 cells. After inhibition of new protein synthesis by cycloheximide treatment, CX50fs fell to nearly undetectable levels after 4 hours, whereas CX50 could still be detected after 18 hours. CX50fs levels were dramatically increased following treatment of cells with epoxomicin (a proteasomal inhibitor) while chloroquine (a lysosomotropic agent) had little effect. Treatment of HeLa cells expressing CX50fs with epoxomicin also resulted in formation of many gap junction plaques and rescued intercellular transfer of Neurobiotin to the levels observed in cells expressing the wild type connexin.
Conclusions:
The CX50fs mutant is rapidly degraded by a proteasome-dependent pathway in mammalian cells. This accelerated degradation leads to the very low abundance of gap junction plaques and levels of intercellular communication in untreated cells expressing this mutant. The epoxomicin-induced rescue of gap junction formation and intercellular communication suggests potential therapeutic approaches to prevent cataract formation using proteasomal inhibitors.
Keywords: 532 gap junctions/coupling •
447 cell-cell communication •
445 cataract