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Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a “higher-NKA-in-hyposmotic media” response in gills of euryhaline milkfish, Chanos chanos

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Abstract

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na+, K+-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1–β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1–β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1–β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1–β1 association in gill ionocytes of euryhaline teleosts.

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Acknowledgements

This study was supported by a grant from the Ministry of Science and Technology (MOST), Taiwan, to T.H. Lee (103-2311-B-005-004-MY3; 104-2911-I-005 -502) and the Taiwan Comprehensive University System (103TCUS03). The monoclonal antibodies α5 and α6F were purchased from the Developmental Studies Hybridoma Bank (DSHB) maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, under Contract N01-HD-6-2915, NICHD, USA.

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  1. Department of Life Sciences, National Chung Hsing University, 145 Xingda Road, Taichung, 402, Taiwan, Republic of China

    Yau-Chung Hu, Keng-Fu Chu, Wen-Kai Yang & Tsung-Han Lee

  2. Bachelor Degree Program in Animal Healthcare, Hungkuang University, Taichung, 433, Taiwan, Republic of China

    Wen-Kai Yang

  3. Agricultural Biotechnology Center, National Chung Hsing University, Taichung, 402, Taiwan, Republic of China

    Tsung-Han Lee

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Communicated by G. Heldmaier.

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360_2017_1066_MOESM2_ESM.tif

Supplementary Fig. S1 Confocal micrographs of the whole-mount milkfish gills showed the background images of gill auto-fluorescence and secondary antibodies. (A) light scope; (B) background level of staining with only the AlexaFluor 546 conjugated goat anti-mouse antibody (for labeling the NKA α subunit in this study; (C) background level of staining with only the AlexaFluor 488 conjugated donkey anti-goat antibody (for labeling the NKA β subunit). F, filament; L, lamella. Scale bar = 50 μm (TIF 7148 KB)

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Hu, YC., Chu, KF., Yang, WK. et al. Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a “higher-NKA-in-hyposmotic media” response in gills of euryhaline milkfish, Chanos chanos . J Comp Physiol B 187, 995–1007 (2017). https://doi.org/10.1007/s00360-017-1066-9

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