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Na⁺+K⁺-ATPase in gills of the blue crab Callinectes sapidus: cDNA sequencing and salinity-related expression of -subunit mRNA and protein

David W. Towle^1,*, Ryan S. Paulsen¹, Dirk Weihrauch¹, Marek Kordylewski¹, Cristina Salvador², Jean-Hervé Lignot³ and Céline Spanings-Pierrot³

¹ Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA and Lake Forest College, Lake Forest, IL 60045, USA,
² Duke University, Durham, NC 27706, USA and
³ Université Montpellier II, 34095 Montpellier Cedex 5, France

View larger version (71K): [in a new window]   Fig. 1. cDNA and predicted amino acid sequence of the Na++K+-ATPase -subunit from gills of the blue crab Callinectes sapidus. Stop codons in the 5' untranslated region (including one in-frame, underlined), the putative stop site and stop codons downstream from the stop site are indicated in red. A Kozak consensus sequence around the likely start codon (underlined) is shown in green. Transmembrane domains predicted by hydrophobicity analysis are shown in blue. The location of the degenerate primers NAK10F and NAK16R, employed in the initial amplification, are indicated by right- and left-pointing arrowheads respectively. The polyadenylation signal in the 3' untranslated region is indicated in violet. GenBank Accession Number AF327439

View larger version (105K): [in a new window]   Fig. 2. Multiple alignment of the Callinectes sapidus -subunit amino acid sequence with examples from other arthropods (Drosophila melanogaster and two isoforms from Artemia franciscana) and from vertebrates (Torpedo californica, Anguilla anguilla, Xenopus laevis, Gallus gallus and Homo sapiens). Alignment was produced using ClustalW and GeneDoc software. Blue, 100 % agreement; green, 80 %; yellow, 60 %. Putative transmembrane domains are indicated by solid black lines; the likely ATP binding site is indicated by a solid red line (Horisberger et al., 1991).

View larger version (39K): [in a new window]   Fig. 3. Estimate of Na++K+-ATPase -subunit mRNA abundance in total RNA extracts of anterior and posterior gills of Callinectes sapidus acclimated for at least 2 weeks to 35 or 5  salinity. Gills 3–5 (anterior) and 6–8 (posterior) were pooled from three individuals for each RNA preparation. mRNA levels were evaluated by duplex quantitative RT-PCR under conditions of limiting template, with arginine kinase mRNA serving as an invariant standard (Kotlyar et al., 2000). The primers employed to amplify Na++K+-ATPase -subunit cDNA were NAK10F and NAK16R and for arginine kinase AKF5 (5'-CGCTGAGTCTAAGAAGGGATT-3') and AKCALLR1 (5'-CCCAGGCTTGTCTTCTTGTCC-3'). Biotinylated PCR products were visualized after 22 cycles using a streptavidin/alkaline phosphatase procedure (Phototope, New England Biolabs). The data are representative of three separate experiments.

View larger version (56K): [in a new window]   Fig. 4. Western blot analysis of Na++K+-ATPase -subunit protein in partially purified plasma membrane preparations from anterior and posterior gills of Callinectes sapidus acclimated for 2 weeks to 35 or 5  salinity. Gills 3–5 (anterior) and 6–8 (posterior) from four crabs were pooled for each treatment. Following SDS–polyacrylamide gel electrophoresis and transfer to nylon membranes, -subunit protein was detected by incubation with 5 monoclonal antibody. The left-hand lane contains Kaleidoscope prestained standards with the indicated molecular masses. The data are representative of three separate experiments.

View larger version (103K): [in a new window]   Fig. 5. Immunocytochemical localization of Na++K+-ATPase -subunit protein in the basolateral membrane of gill epithelial cells in the blue crab Callinectes sapidus. (A) Phase-contrast photomicrograph of portions of two cross-sectioned gill lamellae. c, cuticle; e, epithelial layer; s, intralamellar septum; h, hemolymph space. (B) Immunocytochemical identification of -subunit protein using the 5 monoclonal antibody against a highly conserved cytosolic epitope. Intense fluorescence is evident in the basolateral membrane region (b) but not the apical membrane region (a) of the epithelial cells. Scale bars, 100 µm.