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First published online May 1, 2006
Journal of Experimental Biology 209, 1928-1943 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02190
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Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss)

A. Shahsavarani1, B. McNeill1, F. Galvez2, C. M. Wood2, G. G. Goss3, P.-P. Hwang4 and S. F. Perry1,*

1 Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada
2 Department of Biology, McMaster University, 1280 Main Street W, Hamilton, ON L8S 4K1, Canada
3 Department of Biological Sciences, University of Alberta, Edmonton, Alberta T5G 2E9, Canada
4 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan


Figure 1
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  		Fig. 1. Nucleotide and predicted amino acid sequence of rainbow trout (Oncorhynchus mykiss) gill epithelial calcium channel (ECaC). The sequence shown represents the predicted coding region including the start (underlined) and stop (asterisk) codons. The sequence was obtained by RACE and verified by full-length PCR and cloning.

 

Figure 2
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  		Fig. 2. Predicted rainbow trout (Oncorhynchus mykiss) epithelial calcium channel (ECaC) protein functional domains. Bold-underline indicates possible ankyrin repeats; boxed sequence, ion transport regions; bold sequence within the box, pore-forming region; Asterisks (*), predicted phosphorylation sites: serine (S)=16, threonine (T)=8, tyrosine (Y)=5.

 

Figure 3
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  		Fig. 3. Phylogenetic analysis of rainbow trout (Oncorhynchus mykiss) epithelial calcium channel (ECaC). The phylogenetic tree was constructed using PHYML (maximum likelihood) with support for nodes determined through bootstrapping of 1000 pseudo-datasets. Branches are drawn to scale; the scale bar represents replacement of 5% of the amino acids in the protein alignment. See Table 2 for the description of genes used in this tree; c, chicken; cf, crayfish; d, dog; f, Fugu; h, human; m, mouse; r, rat; rb, rabbit; rt, rainbow trout; x, Xenopus; z, zebrafish.

 

Figure 4
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  		Fig. 4. Phylogenetic analysis of the TRP gene family. The phylogenetic tree was constructed using neighbour-joining algorithms with bootstrap analysis of 100 pseudo-datasets for node support. Scale bar represents replacement of 5% of the amino acid in the protein alignment. See Table 3 for description of genes used in this tree; c, chicken; cf, crayfish; f, Fugu; h, human; m, mouse; rt, rainbow trout; x, Xenopus; z, zebrafish.

 

Figure 5
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  		Fig. 5. (A) Representative and (B) mean tissue distributions of epithelial calcium channel (ECaC) in rainbow trout (Oncorhynchus mykiss) as determined through RT–PCR and real-time RT–PCR, respectively. ECaC-QPCR1 and ECaC-QPCR2 primers were used for PCR detection of ECaC, and ß-actin-FWD and ß-actin-REV primers were used for the detection of ß-actin (see Table 1 for primer sequences). For each tissue, a no template control (NTC) sample was tested (data not shown for real-time PCR results). For real-time PCR, the results are presented as the expression of ECaC relative to ß-actin and standardised to ECaC expression in the gill (N=4).

 

Figure 6
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  		Fig. 6. Localisation of epithelial calcium channel (ECaC), Na+K+-ATPase and nuclei in rainbow trout (Oncorhynchus mykiss) gill epithelia. The cell nuclei appear blue, ECaC appears green and Na+/K+-ATPase appears red; co-localization of ECaC with Na+/K+-ATPase appears yellow. (A) A representative image of a gill section treated with primary antibodies against ECaC and Na+/K+-ATPase. Areas within the boxes illustrate cells exhibiting only ECaC immunoreactivity; arrows indicate cells exhibiting only Na+/K+-ATPase immunoreactivity; asterisks indicate cells exhibiting co-localization of ECaC and Na+/K+-ATPase. (B) A representative image of a gill section pre-absorbed with the ECaC peptide antigen. (C) A representative image of a gill section on which the ECaC primary antibody was omitted. (D) A higher magnification image showing two Na+/K+-ATPase-positive cells with one displaying no ECaC immunoreactivity (1) and the other exhibiting co-localization (2). (F) A representative western blot showing the presence of a single immunoreactive band at 90 kDa in trout gill; the sizes of the protein markers (kDa) are indicated on the left.

 

Figure 7
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  		Fig. 7. Localisation of epithelial calcium channel (ECaC) and Na+/K+-ATPase mRNA and protein in rainbow trout (Oncorhynchus mykiss) gill epithelia. (A) A representative image of a gill section probed with a homologous ECaC oligonucleotide probe: arrows labelled a indicate cells expressing detectable levels of ECaC mRNA; arrows labelled b indicate MRCs expressing detectable levels of ECaC mRNA and those labelled c indicate MRCs that do not appear to express high levels of ECaC mRNA. (B) A representative image of a gill section treated with primary antibodies against ECaC and Na+/K+-ATPase; the cell nuclei appear blue, ECaC appears green and Na+/K+-ATPase appears red; co-localization of ECaC with Na+/K+-ATPase appears yellow; asterisks indicate areas of intense ECaC mRNA or protein at the tips of lamellae where MRCs are rarely found. (C) A representative image of a gill section probed with a homologous Na+/K+-ATPase oligonucleotide probe.

 

Figure 8
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  		Fig. 8. Localisation and quantitative distribution of epithelial calcium channel (ECaC) and Na+/K+-ATPase protein in suspensions of rainbow trout (Oncorhynchus mykiss) gill epithelial cells using (A–E) immunocytochemistry and (F) flow cytometry. In A–E, the cell nuclei appear blue, ECaC appears green and Na+/K+-ATPase appears red; co-localization of ECaC with Na+/K+-ATPase appears yellow. (A,B) Representative images of crude cell suspension prior to separation of different cell types: (1) ECaC-positive cells, (2) Na+/K+-ATPase-positive cells and (3) cells co-expressing ECaC and Na+/K+-ATPase. (C) A representative image from a cell suspension enriched with pavement cells (PVCs). (D,E) Representative images from purified cell suspensions enriched with peanut lectin agglutinin-negative (PNA–) mitochondria rich (MR) cells. (F) The distribution of single- and double-labelled events (cells) as determined by flow cytometry in cell suspensions enriched with PVCs, filled bars; N=4) or PNA– MRCs (unfilled bars; N=4). Data are shown as means ± 1 s.e.m.; *significant difference between the two cell populations (rank-sum test, P<0.05).

 

Figure 9
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  		Fig. 9. Relative epithelial calcium channel (ECaC) mRNA expression in enriched populations of pavement cells (PVCs), peanut lectin agglutinin-negative (PNA–) mitochondria rich (MR) cells and PNA+ MR cells. Data are shown as means ± 1 s.e.m. (N=6).

 

Figure 10
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  		Fig. 10. (A,B) Immunocytochemical localisation of epithelial calcium channel (ECaC) in single-seeded rainbow trout gill epithelial cell culture. The cell nuclei appear blue and ECaC appears green. Note the presence of both ECaC-positive and ECaC-negative (denoted by asterisk) cells. (C) Omission of primary antibody eliminated all fluorescence.

 

Figure 11
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  		Fig. 11. Immunocytochemical localisation of epithelial calcium channel (ECaC) and Na+/K+-ATPase in double-seeded rainbow trout gill epithelial cell cultures using (A) confocal or (B–D) epifluorescence microscopy. In B–D, the cell nuclei appear blue, ECaC appears green and Na+/K+-ATPase appears red; co-localization of ECaC with Na+/K+-ATPase appears yellow. (D) MitoTrackerTM was used to localize mitochondria-rich cells (stained green) and ECaC (red). A is a reconstruction of 22 optical sections showing the presence of ECaC-positive mitochondria rich cells (MRCs, arrows) lying on top of a confluent layer of ECaC-positive and ECaC-negative (asterisks) pavement cells. In B and C the cell types are: (1) ECaC-positive cells; (2) Na+/K+-ATPase-positive cells; (3) cells co-expressing ECaC and Na+/K+-ATPase; (4) cells positive for neither ECaC nor Na+/K+-ATPase. In D, the cell types are: (2) MRCs; (3) MRCs co-expressing ECaC.

 




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