The role of phosphorylation in calmodulin-mediated gating of human AQP0

Biochem J. 2023 Nov 30:BCJ20230158. doi: 10.1042/BCJ20230158. Online ahead of print.ABSTRACTAquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here we express recombinant and functional human AQP0 in P. pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis (MST) and surface plasmon resonance (SPR) technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM-binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM-binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphor...

Source: The Biochemical Journal - November 30, 2023 Category: Biochemistry Authors: Stefan Kreida Jennifer Virginia Roche Julie Winkel Missel Tamim Al-Jubair Carl Johan Hagstr ömer Veronika Wittenbecher Sara Linse Pontus Gourdon Susanna T örnroth-Horsefield Source Type: research