The Arg233Lys AQP0 Mutation Disturbs Aquaporin0-Calmodulin Interaction Causing Polymorphic Congenital Cataract

Shanshan Hu,Yanhua Qi,Hui Lin,Binbin Wang,Edathara Abraham

doi:10.1371/journal.pone.0037637

Abstract

Calmodulin (CaM) directly interacts with the aquaporin 0 (AQP0) C-terminus in a calcium dependent manner to regulate the water permeability of AQP0. We previously identified a missense mutation (p.R233K) in the putative CaM binding domain of AQP0 C-terminus in a congenital cataract family. This study was aimed at exploring the potential pathogenesis of this mutation causative of cataract and mainly identifying how it influenced the binding of AQP0 to CaM. Wild type and R233K mutant AQP0 with EGFP-tag were transfected separately into Hela cells to determine the expression and subcellular localizations. The co-immunoprecipitation (CoIP) assay was used to detect the interaction between AQP0 and CaM. AQP0 C-terminus peptides were synthesized with and without R233K, and the binding abilities of these peptides to CaM were assessed using a fluorescence binding assay. Localizations of wild type and R233K mutant AQP0 were determined from EGFP fluorescence, and the chimeric proteins were both localized abundantly in the plasma membrane. Protein expression levels of the culture cells showed no significant difference between them. The results from CoIP assay implied that R233K mutant presented more weakly in association with CaM than wild type AQP0. The AQP0 C-terminal mutant peptide was found to have 2.5-fold lower binding affinity to CaM than wild type peptide. These results suggested that R233K mutation did not affect the expression, location and trafficking of the protein but did influence the interaction between AQP0 and CaM. The binding affinity of AQP0 C-terminus to CaM was significantly reduced. Due to lack of the modulation of the Ca2+-calmodulin complex, the water permeability of AQP0 was subsequently augmented, which might lead to the development of this cataract.

Highlights

Aquaporins (AQPs) are integral membrane proteins that are ubiquitous transmembrane channels to facilitate the diffusion of water and/or selected small neutral solutes, such as urea and glycerin, across the membrane of the cell [1]
The empty vector pEGFP-n1 was located in both the nucleus and cytoplasm, while both wild type aquaporin 0 (AQP0) (WT-AQP0-EGFP) and R233K mutant AQP0 (R233KAQP0-EGFP) were localized abundantly in the plasma membrane of Human cervical cancer (Hela) cells. These images mainly illustrated that the substitution of lysine for arginine at position 233 in the AQP0 did not impact the trafficking of the expressed protein to the plasma membrane
Representative western blot results showed that wild type AQP0 (WT-AQP0) and R233K mutant AQP0 (R233KAQP0) were all exogenously expressed at comparable levels in comparison with the b-actin loading control (Figure 2)

Summary

Introduction

Aquaporins (AQPs) are integral membrane proteins that are ubiquitous transmembrane channels to facilitate the diffusion of water and/or selected small neutral solutes, such as urea and glycerin, across the membrane of the cell [1]. AQP1 is expressed in lens epithelial cells, while AQP0 is abundant in lens fibers, which contributes to over 50% of total membrane proteins of the fibers [3] These two aquaporins are virtually impermeable to anything but water. The high concentrations of AQP0 are gradually accumulated during the procedure that new lens fibers are continuously differentiated from epithelial cells and subsequently turn into mature lens fibers [5]. It provides the major permeability pathway of water in lens fibers [4]. The lens (particular the center of the lens) relies heavily on this transport system to preserve the normal flow of water across the cells to maintain lens transparency and homeostasis [5]

Objectives

Methods

Results

Conclusion