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. 2018 Sep 7:9:1244.
doi: 10.3389/fphys.2018.01244. eCollection 2018.

Expression of Na+/K+-ATPase Was Affected by Salinity Change in Pacific abalone Haliotis discus hannai

Yanglei Jia  1   2   3 , Xiao Liu  1   2
Affiliations

Expression of Na+/K+-ATPase Was Affected by Salinity Change in Pacific abalone Haliotis discus hannai

Yanglei Jia et al. Front Physiol. .

Abstract

Na+/K+-ATPase (NKA) belongs to the P-type ATPase family, whose members are located in the cell membrane and are distributed in diverse tissues and cells. The main function of the NKA is to regulate osmotic pressure. To better understand the role of NKA in osmoregulation, we first cloned and characterized the full-length cDNAs of NKA α subunit and β subunit from Pacific abalone Haliotis discus hannai in the current study. The predicted protein sequence of the NKA α subunit, as the catalytic subunit, was well conserved. In contrast, the protein sequence of the β subunit had low similarity with those of other species. Phylogenetic analysis revealed that both the α and β subunits of the NKA protein of Pacific abalone were clustered with those of the Gastropoda. Then, the relationship between salinity changes and the NKA was investigated. Sudden salinity changes (with low-salinity seawater (LSW) or high-salinity seawater (HSW)) led to clear changes in ion concentration (Na+ and K+) in hemolymph; however, the relative stability of ion concentrations in tissue revealed that Pacific abalone has a strong osmotic pressure regulation ability when faced with these salinity changes. Meanwhile, the expression and activity of the NKA was significantly decreased (in LSW group) or increased (in HSW group) during the ion concentration re-establishing stages, which was consistent with the coordinated regulation of ion concentration in hemolymph. Moreover, a positive correlation between cyclic adenosine monophosphate (cAMP) concentrations and NKA mRNA expression (NKA activity) was observed in mantle and gill. Therefore, the sudden salinity changes may affect NKA transcription activation, translation and enzyme activity via a cAMP-mediated pathway.

Keywords: Na+/K+-ATPase; cAMP; ion transportation; osmoregulation; salinity.

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Figures

FIGURE 1
FIGURE 1
Experimental design. (A) Treatment and sampling time points. (B) Application of sampled tissue.
FIGURE 2
FIGURE 2
Concentrations of Na+ and K+ ions. (A) The correlation between salinity and concentrations of Na+ and K+. (B) The concentration of Na+ in hemolymph after the sudden salinity change at different times. (C) The concentration of K+ in hemolymph after the sudden salinity change at different times. (D) The concentration of Na+ in mantle and gill after the sudden salinity change at different times. (E) The concentration of K+ in mantle and gill after the sudden salinity change at different times. Different letters on data indicate statistically significant differences between the results (P < 0.05). n = 4/time-point.
FIGURE 3
FIGURE 3
The cDNA sequence and deduced amino acid sequence of the Na+/K+-ATPase α subunit from H. discus hannai (Accession number: MG767304). The start codon (ATG) is boxed in green. The asterisk (∗) indicates the stop codon. The P-type ATPase motif is indicated by a blue box. A polyadenylation signal sequence AATAAA is circled with a red oval. The transmembrane regions are underlined with green and numbered (1–10).
FIGURE 4
FIGURE 4
The cDNA sequence and deduced amino acid sequence of the Na+/K+-ATPase β subunit from H. discus hannai (Accession number: MG767305). The start codon (ATG) is boxed in green. The asterisk (∗) indicates the stop codon. A polyadenylation signal sequence AATAAA is circled with a red oval. The transmembrane region is indicated with green underline.
FIGURE 5
FIGURE 5
Multiple alignment of the HdNKA α subunit sequence with NKA α subunit sequences deposited in GenBank. The amino acids highlighted in gray are identical in all sequences. The amino acids highlighted in pink are conserved more than 75%. The amino acids highlighted in cyan are conserved more than 50%. The regions underlined with green and numbered (1–10) are transmembrane regions.
FIGURE 6
FIGURE 6
Multiple alignment of the HdNKA β subunit sequences with NKA β subunit sequences deposited in GenBank. The amino acids highlighted in gray are identical in all sequences. The amino acids highlighted in pink are conserved more than 75%. The amino acids highlighted in cyan are conserved more than 50%.
FIGURE 7
FIGURE 7
Phylogenetic tree, transmembrane structure and expression profile analysis. (A) Phylogenetic tree analysis of HdNKA α subunit from Pacific abalone and NKA α subunits from other species. (B) Phylogenetic tree analysis of HdNKA β subunit from Pacific abalone and NKA β subunits from other species. (C) Topology model of the α subunit of HdNKA. The yellow spheroids represent the cell membrane, and the blue rod-like structure represents the transmembrane region. The numbers in the rod represent the amino acid sites. (D) Topology model of the HdNKA β subunit. The yellow spheroids represent the cell membrane. The red rod and oval structures represent the transmembrane region and highly glycosylated extracellular domain, respectively. The numbers in the rod represent the amino acid sites. (E) Transcript levels of Hdnka α subunit in the tissues obtained from Pacific abalone. (F) Transcript levels of Hdnka β subunit in the tissues obtained from Pacific abalone. Different letters on data indicate statistically significant differences between the results (P < 0.05). n = 4/time-point.
FIGURE 8
FIGURE 8
Expression and activity analysis of Na+/K+-ATPase α and β subunits. (A) Influence of salinity changes on the mRNA level of the Hdnka α subunit in mantle. (B) Influence of salinity changes on the mRNA level of the Hdnka β subunit in mantle. (C) Influence of salinity changes on the mRNA level of the Hdnka α subunit in gill. (D) Influence of salinity changes on the mRNA level of the Hdnka β subunit in gill. (E) Influence of salinity changes on the activity of HdNKA in mantle. (F) Influence of salinity changes on the activity of HdNKA in gill. (G) Influence of salinity changes on the expression of the HdNKA α subunit in mantle determined with western blot. (H) Influence of salinity changes on the expression of the HdNKA α subunit in gill determined with western blot. Different letters on data indicate statistically significant differences between the results (P < 0.05). n = 4/time-point.
FIGURE 9
FIGURE 9
cAMP concentrations and correlation analysis. (A) Influence of salinity changes on the concentration of cAMP in mantle. (B) Influence of salinity changes on the concentration of cAMP in gill. (C) Correlation analysis between cAMP concentration and Hdnka α and β subunit expression in mantle. (D) Correlation analysis between cAMP concentration and Hdnka α and β subunit expression in gill. (E) Correlation analysis between cAMP concentration and HdNKA activity in mantle. (F) Correlation analysis between cAMP concentration and HdNKA activity in gill. Different letters on data indicate statistically significant differences between the results (P < 0.05). n = 4/time-point.
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