Increased Association of Deamidated αA-N101D with Lens membrane of transgenic αAN101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization

We have generated two mouse models, in one by inserting the human lens alpha AN101D transgene in CRY alpha A(N101D) mice, and in the other by inserting human wild-type alpha A-transgene in CRY alpha A(WT) mice. The CRY alpha A(N101D) mice developed cortical cataract at about 7-months of age relative to CRY alpha A(WT) mice. The objective of the study was to determine the following relative changes in the lenses of CRY alpha A(N101D)- vs. CRY alpha A(WT) mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of alpha A(N101D) vs. WT alpha A proteins, and changes in intracellular ionic imbalance and membrane organization.MethodsLenses of varying ages from CRY alpha A(WT) and CRY alpha A(N101D) mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the alpha AN101D- and WT alpha A proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant alpha A(N101D)- and WT alpha A proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.ResultsCompared to the lenses of CRY alpha A(WT), the lenses of CRY alpha A(N101D) mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of alpha AN101D- relative to WT alpha A protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRY alpha A(N101D) lenses. (C) During in vitro assay, the greater levels of binding alpha AN101D- relative to WT alpha A protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRY alpha A(N101D-) than those of CRY alpha A(WT) mice.ConclusionsThe results show that an increased lens membrane association of alpha A(N101D)-(-)relative WT alpha A protein in CRY alpha A(N101D) mice than CRY alpha A(WT) mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.