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Involvement of acid-sensing ion channels in Na+ uptake in freshwater fish Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Dymowska, Agnieszka K
Supervisor and department
Goss, Greg (Biological Sciences)
Examining committee member and department
Chang, John (Biological Sciences)
Brauner, Colin (Department of Zoology)
Ali, Declan (Biological Sciences)
Cheeseman, Chris (Department of Physiology)
Department of Biological Sciences
Physiology, Cell and Developmental Biology
Date accepted
Graduation date
Doctor of Philosophy
Degree level
The molecular identity of the Na+ uptake mechanism across the gills of freshwater fish has been the subject of lively debate for decades. Despite the extensive evidence for NHE mediated Na+ uptake, thermodynamic constraints on the function of NHEs at low ion concentrations and low environmental pHs suggest that other mechanisms may need to exist in fish species inhabiting such environments. An alternative mechanism, whereby Na+ enters through an epithelial Na+ channel (ENaC) was previously proposed. However, efforts to identify ENaC homologues in teleost fishes have not been successful and therefore, alternatives to ENaC needed to be explored. In this thesis, I investigated a possible role for acid-sensing ion channels (ASICs), which are close relatives to ENaCs, to serve as epithelial channels for Na+ uptake by the gill of freshwater rainbow trout and zebrafish. I cloned asic1 and asic4 homologues in the rainbow trout and demonstrated that they are expressed in the gills and isolated mitochondrion-rich cells. Moreover, I demonstrated that six asic subunits, asic1.1, 1.2, 1.3, 2, 4.1, and 4.2, are present in the zebrafish gills. Immunohistochemical analysis using a custom made anti-zASIC4.2 antibody was conducted for both zebrafish adult and larvae, and for rainbow trout. In rainbow trout, double staining with anti-ASIC4.2 and anti-NKA antibodies demonstrated that ASIC4 localizes to NKA-rich MRCs in the gill, whereas in zebrafish adult and larvae, staining with anti-ASIC4.2, anti-VHA, and anti-NKA antibodies and ConcanavalinA revealed that ASIC4.2 localizes to HR MRCs. Furthermore, confocal microscopy demonstrated that in both species, ASIC is found in the apical region of the MRCs. Pharmacological inhibitors of ASICs decreased Na+ uptake in adult rainbow trout and zebrafish in a dose-dependent manner. Moreover, knock-down of ASIC4.2 with morpholino oligonucleotide resulted in reduced Na+ uptake in the morphant zebrafish larvae reared in low Na+ and low pH medium. Based on the findings in my thesis, I present a revised model for Na+ uptake in freshwater fish, whereby ASIC4 is proposed as one possible mechanism for Na+ acquisition in rainbow trout and zebrafish. This is the first demonstration of an apical Na+ channel in the gills of fishes and resolves a long-standing dispute about the identity of a channel-mediated Na+ uptake mechanism. Moreover, in a broader context, this is the first demonstration of ASICs being expressed in an epithelia tissue in any vertebrate species, which expands the functional role for this family of channels.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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