The Defective AQP0 of the CatFR Mutant Mice (AQP0–LTR) Alters Water Permeability and Calcium Regulation Of Wild Type AQP0

Abstract

Abstract: : Purpose: Investigate at the molecular level the interaction of AQP0 and AQP0–LTR expressed together in three different expression systems. Methods: Histology and immunohistochemistry of lenses from transgenic Cat^Fr mice expressing wild type AQP0 (Tg–AQP0), measurement of water permeability in injected oocytes, visualization of trafficking in transfected human lens epithelial cells (B3). Results: AQP0–LTR interacts with AQP0 and alters its biophysical properties. Lenses of Tg–AQP0 show improved organization of fiber cells in the outer cortex but harbor amorphous cells in the lens nucleus. Furthermore, two exogenous systems expressing various ratios of AQP0 and AQP0–LTR revealed that AQP0–LTR alters the function of AQP0 in different ways depending on the quantity of AQP0–LTR. When 10 ng of AQP0 and 2.5 ng of AQP0–LTR cRNA are injected into oocytes, the water permeability is reduced by a factor of two compared with 10 ng of AQP0 cRNA only. pH and Ca²⁺ regulation remain intact. When 1 µg of AQP0 and 0.25 µg of EGFP–AQP0–LTR DNA are transfected in B3 cells, the EGFP–tagged AQP0–LTR appears in the plasma membrane. When 10 ng of both AQP0 and AQP0–LTR cRNA are injected into oocytes, the water permeability is the same as that of oocytes injected with AQP0 only, but Ca²⁺ regulation of water permeability is abolished. In B3 cells transfected with equal levels (1µg) of AQP0 and EGFP–AQP0–LTR, no EGFP tagged AQP0–LTR is observed in the plasma membrane. Conclusions: The molecular nature of the dominant negative phenotype of Cat^Fr mutation arises from interference of AQP0–LTR with the normal function of AQP0 when they are expressed in the same cell. This interaction may contribute to the early termination of fiber cell growth in Tg–AQP0 mice. AQP0–LTR produces a gain of function mutation, which depends on the amount of AQP0–LTR, and which reduces the water permeability or eliminates the calcium regulatory pathway of wild type AQP0. These effects probably contribute to the dominant inheritance pattern of the Cat^Fr cataract.

Keywords: cataract • transgenics/knock-outs • cell membrane/membrane specializations