Characterization of Eukaryotic Aquaporin Regulation

Abstract

Aquaporins, as transmembrane proteins essential for cellular water balance maintenance, are increasingly recognized for their modulation by protein-protein interactions, often implicated in various human pathologies. This thesis explores the function and regulation of aquaporins in human, plant and fish, focusing on human aquaporin 4 (hAQP4), spinach aquaporin (SoPIP2;1) and climbing perch aquaporin (cpAQP1aa), which function in different environments with different osmolarity challenges. Employing bimolecular fluorescence complementation and flow cytometry in vivo, we established standardized sample preparation methods, distinguishing constructive interactions from randomly formed complexes. This approach, utilized across various studies, included screening for the interaction of the key regulator CaM with human aquaporins as well as screening a human brain expression library for interaction with hAQP4. By employing FACS and an established sorting gate to capture strong fluorescence signals, we efficiently isolated novel interaction candidates to water channel in the brain. While extensive insight into the structural gating mechanism of SoPIP2;1 was gained through X-ray crystallography, reproducing such crystal quality for other membrane proteins remains challenging. Hence, alternative techniques, such as continuous diffraction, were explored to obtain structural data. Furthermore, this thesis delves into the molecular mechanism of AQP1 from the fish Anabas testudineus. Through structural, mutational and functional studies, key residues responsible for the osmoregulatory mechanisms have been discovered. Integration of stop-flow assays and molecular dynamics simulations revealed a previously unknown extracellular gating mechanism for this aquaporin isoform involving phosphorylation. Altogether, the work summarized here presents a new robust method for evaluation and screening of membrane protein complexes, with potential importance for regulation. Furthermore, high-resolution structures are unbeatable tools for analysing protein function, and in this thesis, alternative approaches as well as the first structure of an aquaporin from fish are presented, shedding new light on eukaryotic aquaporin regulation.