the arg233lys aqp0 mutation disturbs aquaporin0-calmodulin interaction causing polymorphic congenital cataract的arg233lys aqp0突变干扰aquaporin0-calmodulin交互造成多态先天性白内障.pdfVIP

The Arg233Lys AQP0 Mutation Disturbs Aquaporin0- Calmodulin Interaction Causing Polymorphic Congenital Cataract 1 2 1 1 Shanshan Hu , Binbin Wang , Yanhua Qi *, Hui Lin * 1 Department of Ophthalmology, the 2nd Affiliated Hospital of Harbin Medical University, Harbin, China, 2 Center for Genetics, National Research Institute for Family Planning, Beijing, China 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