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

ABSTRACT: We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA(N101D) mice, and in the other by inserting human wild-type αA-transgene in CRYαA(WT) mice. The CRYαA(N101D) mice developed cortical cataract at about 7-months of age relative to CRYαA(WT) mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA(N101D)- vs. CRYαA(WT) mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA(N101D) vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. METHODS: Lenses of varying ages from CRYαA(WT) and CRYαA(N101D) mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA 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 αA(N101D)- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca(2+) uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. RESULTS: Compared to the lenses of CRYαA(WT), the lenses of CRYαA(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 αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαA(N101D) lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca(2+) uptake were observed in cultured lens epithelial cells of CRYαA(N101D-) than those of CRYαA(WT) mice. CONCLUSIONS: The results show that an increased lens membrane association of αA(N101D)-(−)relative WTαA protein in CRYαA(N101D) mice than CRYαA(WT) mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.