Submitted on May 29, 2006
Accepted on September 7, 2006
Molecular characterization of water selective AQP, EbAQP4, in hagfish; insight into ancestral origin of AQP4
Goro Nishimoto1*, Go Sasaki2, Eishin Yaoita1, Masaaki Nameta1, Huiping Li3, Kyoko Furuse4, Hidehiko Fujinaka1, Yutaka Yoshida1, Akihisa Mitsudome5, and Tadashi Yamamoto1
1 Department of Structural Pathology, Institute of Nephrology, Niigata, Japan
2 Biohistory Research Hall, JT, Japan
3 Department of Structural Pathology, Institute of Nephrology, Japan
4 KAN Research Institute, Japan
5 Dept. of Pediatrics, Fukuoka University School of Medicine, Japan
* To whom correspondence should be addressed. E-mail: gorni{at}med.niigata-u.ac.jp.
Hagfish are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the Mitochondria rich (MR) 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 homologue exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP, 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 8-fold compared to water-injected controls and was not reversibly inhibited by 0.3 mM HgCl2. EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the C-terminus of EbAQP4 also detected (Western blot) a major ~31-kDa band in the gill. Immunohistochemistry and immuno-electron 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.
