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Am J Physiol Regul Integr Comp Physiol 292: R644-R651, 2007. First published September 21, 2006; doi:10.1152/ajpregu.00362.2006
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WATER AND ELECTROLYTE HOMEOSTASIS

Molecular characterization of water-selective AQP (EbAQP4) in hagfish: insight into ancestral origin of AQP4

¹Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata; ²Japan Tobacco, Inc., Biohistory Research Hall, Osaka; ³Knowledge, Action, Network Research Institute, Kyoto; and ⁴Department of Pediatrics, School of Medicine, Fukuoka University, Fukuoka, Japan

Submitted 29 May 2006 ; accepted in final form 7 September 2006

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 HgCl₂. 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 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.

mitochondria-rich cells; orthogonal arrays; pavement cell