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

• Author / Creator

  Dymowska, Agnieszka K

• 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.

• Subjects / Keywords

  • Sodium
  • ASICs
  • Freshwater
  • Ionoregulation
  • Gill

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R31C1TM7Q

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.