Regulation of Aquaporins by Proteins, Metal ions and Phosphate groups

Abstract: Aquaporins (AQPs) are membrane-bound intrinsic water channels that allow the passive diffusion of water and small solutes across cellular membranes. There are 13 highly conserved isoforms of these channels in human, which differ in specificity, regulation and localisation in cells and tissues. The work in this thesis focuses on AQP0 and AQP2; water-specific aquaporins that are known to interact with regulatory proteins via their respective C-termini and which both contain multiple phosphorylation sites within this region. AQP0 is the main aquaporin of the eye lens. It binds calmodulin (CaM); the ubiquitous calcium-sensing protein, and this interaction has been proposed to block the water pore. Using microscale thermophoresis and fluorescence anisotropy we characterised the interaction between CaM and AQP0 in different phosphorylation states. Our results show that CaM binds full-length AQP0 with positive cooperativity, which is not seen when AQP0 C-terminal peptides are used. Moreover, the phospho-mimicking mutants AQP0-S229D and S235D blocks binding to CaM, whereas S231D can bind with similar affinity as wild type but in a different way. Functional water conductance assays confirm previous studies that CaM blocks the channel in a calcium-dependent manner whereas all mutants are insensitive to CaM/Ca and locked in an open state. Using a combination of fluorescence anisotropy, chemical crosslinking and small-angle X-ray scattering, we further show that zinc binding induces a conformational change in CaM, thereby affecting its interaction with AQP0. This may be of physiological importance for CaM-mediated regulation of AQP0 in the eye. AQP2 is regulated by vasopressin-dependent trafficking in the kidney collecting duct, which involves phosphorylation of the AQP2 C-terminus. Here we investigate how phosphorylation affects the interaction with LIP5, an ESCRT-III/Vps4 adaptor protein which plays a role in targeting AQP2 to multivesicular bodies (MVB) and subsequent lysosomal degradation. Our results from far-western blot and MST show that the phospho-mimicking mutants S256E, S261E, T269E and S256E-T269E impair LIP5 binding, whereas S264E does not significantly affect the affinity to LIP5. Removal of all phosphorylation sites through truncation while retaining the LIP5 binding site reduced the affinity further, suggesting that phosphorylation of AQP2 allosterically controls its interaction with LIP5. Our results fit well with the suggested roles of the respective phosphorylation sites in AQP2 trafficking and suggests that LIP5 may have a more direct role in MVB cargo sorting than previously understood.