Identification of Molecular and Functional Mechanisms behind Cell Volume Regulation in Astrocytes

Aquaporin 4 (AQP4) is a highly conserved protein in mammals, since point mutation usually lead to a reduce water permeability. A recent study investigated the effect of a mutation in AQP4 gene in AQP4 membrane protein expression and water permeability. Chloride channels are also involved in the maintenance of homeostasis in the brain and in particular, volume-regulated anion channels (VRAC) mainly address this task. VRACmolecular identity, partially solved in 2014 consist of an essential sub-unit termed as leucine rich repeat containing 8 family member A. Hence, the aim of this study was to investigate the molecular and functional interactions between the mutant D184E of aquaporin 4 protein, with the ion channels TRPV4 and VRAC. Moreover, three antibody raised against LRRC8A antigenic polypeptides have been produced to investigate the expression of VRAC underpinning subunits in cortical astrocytes and mouse brain. The main findings and contribution to theory of my PhD project are the following: 1-the functional antagonism between TRPV4 and VRAC in a heterologous system, previously reported in HEK-293 cells (98), can be overcome by co-expression of AQP4 and TRPV4. 2-The evidence that a D184E mutation of the M1 isoform of AQP4 gene impair the molecular interaction between TRPV4 and AQP4. 3-The definition of the expression pattern of LRRC8A subunits, underpinning VRAC conductance (REF), in COS-7 heterologous system, in astrocytes and in the mouse brain. In conclusion, the results of my PhD studies provide new insights into key molecular and functional player involved central nervous system homeostatic volume regulation. In particular they suggested and reinforce the tenet that a macromolecular complex or a straight functional interaction between TRPV4, AQP4 and VRAC might be essential for the cell volume regulation.