title: Molecular characterization of water-selective AQP (EbAQP4) in hagfish: insight into ancestral origin of AQP4.
abstract: Hagfish (Eptatretus burgeri) are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the mitochondria-rich cells show orthogonal arrays of particles (OAPs) in the gill of hagfish, a known ultrastructural morphology of aquaporin (AQP) in mammalian freeze-replica studies, suggesting that an AQP homolog exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP [E. burgeri AQP4 (EbAQP4)] encodes 288 amino acids, including two NPA motifs and six transmembrane regions. The deduced amino acid sequence of EbAQP4 showed high homology to mammalian and avian AQP4 (rat, 44%; quail, 43%) and clustered with AQP4 subsets by the molecular phylogenetic tree. The osmotic water permeability of Xenopus oocytes injected with EbAQP4 cRNA increased eightfold compared with water-injected controls and was not reversibly inhibited by 0.3 mM HgCl(2). EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the COOH terminus of EbAQP4 also detected (by Western blot analysis) a major approximately 31-kDa band in the gill. Immunohistochemistry and immunoelectron microscopy showed EbAQP4 localized along the basolateral membranes of gill pavement cells. In freeze-replica studies, OAPs were detected on the protoplasmic face of the split membrane comprising particles 5-6 nm long on the basolateral side of the pavement cells. These observations suggest that EbAQP4 is an ancestral water channel of mammalian AQP4 and plays a role in basolateral water transport in the gill pavement cells.
authors.affiliation: Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. gorni@med.niigata-u.ac.jp
authors.forename: Goro
authors.initials: G
authors.lastname: Nishimoto
authors.forename: Go
authors.initials: G
authors.lastname: Sasaki
authors.forename: Eishin
authors.initials: E
authors.lastname: Yaoita
authors.forename: Masaaki
authors.initials: M
authors.lastname: Nameta
authors.forename: Huiping
authors.initials: H
authors.lastname: Li
authors.forename: Kyoko
authors.initials: K
authors.lastname: Furuse
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Fujinaka
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yoshida
authors.forename: Akihisa
authors.initials: A
authors.lastname: Mitsudome
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005614:Freeze Fracturing; D005880:Gills / Q000378:metabolism; D006196:Hagfishes / Q000378:metabolism*; D007150:Immunohistochemistry; D008628:Mercury / Q000494:pharmacology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D010802:Phylogeny; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014867:Water / Q000378:metabolism; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D014867:Water; D012313:RNA; D008628:Mercury