Mechanism Underlying Congenital Cataract Caused By Connexin46Mutation G143R

Connexin (Cx) are integral membrane proteins, which formed in cytoplasm and transfer to cell membrane. Six connexin proteins form connexons, also called hemichannels, which mediate communication between cells and extracellular environment. Gap junctions are cluster of transmembrane channels, which formed by two connexons from adjacent cells. On the cell surface, gap junctions assemble and form gap junction plaque. Gap junction channels allow passage of small molecules (Mr≤1kDa) in contacting cells.In lens, intercellular communication via gap junction and hemichannel is crucial in maintaining osmotic and metabolic homeostasis and ultimately, transparency of the lens. It has known that the mutations of lens fiber connexin (Cx46and Cx50) are associated with congenital cataracts in human. So far, over20mutations of Cx46have been found in congenital cataracts from different origins. Mutations of Cx46could affect the formation and function of gap junction channels and/or hemichannels and lead to cataracts.The Gly143Arg missense mutation on GJA3(Cx46) gene is associated with congenital Coppock cataracts in a Chinese family, which is the first time reported that the mutation of Cx46located in cytoplasmic loop. However, the underlying molecular mechanism has been not characterized.The glycine at position143in Cx46is highly conserved across different animal species and different connexins in human. Exogenous expression of Cx46G143R mutant in HeLa cells revealed similar cellular distribution as wild type Cx46. The Cx46G143R also can form gap junction plaque in HeLa cells.However, by using scrap loading dye transfer assay and microinjection dye transfer method, we found that this mutant decreased gap junctional coupling. In addition, this mutation acted in a dominant negative manner when co-transfected with wild type Cx46. Similarly, when expressed Cx46G143R in paired Xenopus oocytes, this mutation blocked electrical coupling in a dominant negative manner. Triton X-100extraction experiment was used to test the level of gap junction plaque. Western blot result showed that this mutation expressed significantly more Triton X-100insoluble Cx46, when compared with wild type Cx46. We also used dye uptake assay to test the function of hemichannels formed by Cx46G143R. In contrast to its effect on gap junction function, this mutant increased hemichannel activity, even in presence of Ca2+, which reversely correlated with the level of Cx46on the cell surface.The three-dimensional structure prediction by Swiss Model showed that this mutation may change the secondary structure of intracellular loop of Cx46protein. A helical structure located in the intracellular loop was found in Cx46G143R protein. The GST-Cx46loop/Cx46G143R loop fusion was prepared and reacted with C terminal of Cx46by Pull-down experiment. The result showed that Cx46G143R enhanced the interaction between intracellular loop and C terminal of Cx46, when compared with wild type Cx46.On the other side, the mutation Cx46G143R decreased cell viability, increased cell apoptosis and attenuated the resistibility to oxidative stress. And dye uptake assay result showed that the role of this mutation in cell apoptosis was related with increased hemichannel activity.Together these data showed that Cx46G143R mutation could lead to congenital cataract in human with decreasing gap junction function, enhancing hemichannel activity and reducing cell viability, increasing cell apoptosis. This clearly indicated intercellular communication mediated by gap junction is required for maintaining lens transparency by creating the microcirculation of nutrients and metabolites.