AQUA(GLYCERO)PORINS · Integrated analyses of aquaporin structure and function

Final Activity Report Summary - AQUA(GLYCERO)PORINS (Integrated analyses of aquaporin structure and function)

The project has delivered results concerning concepts and techniques from molecular cell biology; physiology, structural biology, computational biology via molecular dynamics simulation, systems biology, organic chemistry and combinatorial chemistry. The following section describes the most outstanding finding during the project period. Firstly, a general mechanism for the solute permeation and selectivity of aquaporins has been established. In this mechanism, hydrophobic regions near the highly conserved NPA motifs, located at the middle of the channel, are the main water rate limiting barriers. In addition, the aromatic/arginine region, the most constricted part of the pore, is the main selectivity filter for the permeation of other uncharged solutes like glycerol, ammonia or urea. During the project period several aquaglyceroporin have been produced. The purified proteins have been used for structural analyses. One result was the high-resolution X-ray structure of the yeast aquaporin. The major scientific finding was that this yeast aquaporin is a gated aquaporin, and a new structural mechanism was discovered. Furthermore, the structure of human AQP5 was determined to a resolution of 2.0Å. This high resolution structure gives novel insights into the role of the central pore - which was occupied by a lipid in the structure. Several new regulatory mechanisms for aquaporins have been discovered. First, the yeast aquaporin is suggested to be regulated by both phosphorylation and mechanosensensitive. Second, voltage-regulated water flux was observed through AQP1 and AQP4 in silico. Furthermore, a mathematical model of AQP2 trafficking and regulation in collecting duct cells was proposed. Our model includes the vasopressin receptor, adenylate cyclase, protein kinase A, and activated as well as inactivated forms of AQP2. To model the chemical reactions we used ordinary differential equations. Through generating, testing and ranking different versions of the model we showed, that some model versions can describe the data equally well as soon as important regulatory parts such as the shutdown of the signal by internalization of the vasopressin-receptor or the negative feedback loop through phosphodiesterases are included. We investigated through time dependent sensitivity analysis, which reactions have the most influence on cAMP and AQP2 in the membrane over time. We predicted the time courses for the involved species at different concentrations, compared them with newly generated data and discussed the competencies of the model. The background for this model was that in healthy individuals, dehydration of the body leads to release of the hormone vasopressin from the pituitary, via the bloodstream, vasopressin reaches the collecting duct cells in the kidney, where the water channel AQP2 is expressed. After stimulation of the vasopressin V2 receptor, intracellular AQP2-containing vesicles fuse with the apical plasma membrane of the collecting duct cells. This leads to increased water reabsorption from the pro-urine into the blood and therefore to enhanced retention of water within the body. Interactions of aquaporins with other molecules such as proteins and lipids have been investigated. First, we studied the formation and stability of the AQP2-LIP5 complex (aquaporin-protein interactions), which is a crucial process during the internalisation of AQP2 from the membrane. Second, we investigated the localisation of DMPC lipids around AQP0, which is an excellent model to study protein-lipid interactions. Several compounds have been identified and successfully tested as potential AQP blockers. An automated computational protocol, combining molecular dynamics simulations, has been fully implemented. This theoretical approach has been integrated to experimental functional assays and organic chemistry synthesis techniques to efficiently design novel specific aquaporin blockers.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз