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

UNLABELLED

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA mice, and in the other by inserting human wild-type αA-transgene in CRYαA mice. The CRYαA mice developed cortical cataract at about 7-months of age relative to CRYαA mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA- vs. CRYαA mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.

METHODS

Lenses of varying ages from CRYαA and CRYαA 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- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca 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, the lenses of CRYαA 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 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 uptake were observed in cultured lens epithelial cells of CRYαA than those of CRYαA mice.

CONCLUSIONS

The results show that an increased lens membrane association of αA-relative WTαA protein in CRYαA mice than CRYαA mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.