Positively charged amino acid residues in the extracellular loops A and C of lens aquaporin 0 interact with the negative charges in the plasma membrane to facilitate cell-to-cell adhesion

Abstract

This investigation was undertaken to find out whether the positive charges in the Extracellular Loops A (ELA) and C (ELC) of Aquaporin 0 (AQP0) are involved in lens fiber cell-to-cell adhesion (CTCA), and the possible mechanism of CTCA. AQP0 ELA or ELC was substituted with the corresponding AQP1 loop via Polymerase Chain Reaction. Positively charged arginine (R) and histidine (H) of mouse AQP0 ELA and ELC were substituted individually with glutamine (Q) to create R33Q, H40Q, R113Q and H122Q by mutagenesis. cRNA expression, immunostaining, Förster Resonance Energy Transfer (FRET) studies and protein analyses showed localization of all mutants except AQP0-AQP1ELC chimera (AQP0 ELC substituted with AQP1 ELC) at the plasma membrane. Osmotic Swelling Assay revealed comparable water permeability (Pf) among AQP0-AQP1ELA, R33Q, R113Q, and WT. CTCA assay demonstrated a significant reduction in adhesion in all mutants compared to the WT (14–73%) suggesting the importance of the conserved positively charged residues of ELA and ELC for adhesion. Studies involving AQP0-transfected L-cells, and lipid vesicles indicated that CTCA was due to the electrostatic interaction between the positively charged amino acids of AQP0 extracellular loops and the negative charges of the plasma membrane. Schematic models are provided to illustrate the mechanism.