title: D184E mutation in aquaporin-4 gene impairs water permeability and links to deafness.
abstract: Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness.
authors.affiliation: Department of General and Environmental Physiology and Center of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy. p.nicchia@biologia.uniba.it
authors.forename: G P
authors.initials: GP
authors.lastname: Nicchia
authors.forename: R
authors.initials: R
authors.lastname: Ficarella
authors.forename: A
authors.initials: A
authors.lastname: Rossi
authors.forename: I
authors.initials: I
authors.lastname: Giangreco
authors.forename: O
authors.initials: O
authors.lastname: Nicolotti
authors.forename: A
authors.initials: A
authors.lastname: Carotti
authors.forename: F
authors.initials: F
authors.lastname: Pisani
authors.forename: X
authors.initials: X
authors.lastname: Estivill
authors.forename: P
authors.initials: P
authors.lastname: Gasparini
authors.forename: M
authors.initials: M
authors.lastname: Svelto
authors.forename: A
authors.initials: A
authors.lastname: Frigeri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051401:Aquaporin 4 / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D003638:Deafness / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010539:Permeability; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D014867:Water / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D014867:Water