Identification of FXYD Protein Genes in a Teleost
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CORE Metadata, citation and similar papers at core.ac.uk Provided by University of Southern Denmark Research Output Am J Physiol Regul Integr Comp Physiol 294: R1367–R1378, 2008. First published February 6, 2008; doi:10.1152/ajpregu.00454.2007. Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change Christian Kølbæk Tipsmark Institute of Biology, University of Southern Denmark, Odense, Denmark Submitted 26 June 2007; accepted in final form 1 February 2008 Tipsmark CK. Identification of FXYD protein genes in a teleost: developmental- and tissue-specific manner, suggesting partic- tissue-specific expression and response to salinity change. Am J ular roles during development or coupled to specific physio- Physiol Regul Integr Comp Physiol 294: R1367–R1378, 2008. First logical functions (10, 40). The FXYD proteins are, in many published February 6, 2008; doi:10.1152/ajpregu.00454.2007.—It is cases, expressed in a tissue-specific manner (6, 12, 15), and, increasingly clear that alterations in Naϩ-Kϩ-ATPase kinetics to fit along the collecting duct of the rat kidney, FXYD isoform Downloaded from the demands in specialized cell types is vital for the enzyme to execute expression is differentiated among segments and cell types (41, its different physiological roles in diverse tissues. In addition to 48). Specific interactions with FXYD proteins may, therefore, tissue-dependent expression of isoforms of the conventional subunits, ϩ ϩ ␣ and , auxiliary FXYD proteins appear to be essential regulatory add to the versatility of the Na -K -ATPase in various tissues, components. The present study identified genes belonging to this and they can be regarded as tissue-specific modulators of ion family in Atlantic salmon by analysis of expressed sequence tags. transport. In addition, FXYD1, FXYD2, and FXYD10 also Based on the conserved domain of these small membrane proteins, seem to operate as regulatory subunits responsive to exterior ϩ ϩ http://ajpregu.physiology.org/ eight expressed FXYD isoforms were identified. Phylogenetic analy- signals, since the interaction with Na -K -ATPase in these iso- sis suggests that six isoforms are homologues to the previously forms is altered by PKA and PKC phosphorylation (18, 25, 43). identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, Little is known about the presence and function of FXYD while two additional isoforms were found (FXYD11 and FXYD12). proteins in nonmammalian vertebrates. In spiny dogfish (Squa- Using quantitative PCR, tissue-dependent expression of the different lus acanthias), a FXYD protein has been characterized and isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, cloned (FXYD10, Ref. 44). In teleosts, three FXYD isoforms and liver. Two isoforms were expressed in several tissues (FXYD5 were identified among expressed sequence tags (ESTs) from and FXYD9), while six isoforms were distributed in a discrete zebrafish by Sweadner and Rael (55). In several teleost species, manner. In excitable tissues, two isoforms were highly expressed in ϩ tissue-dependent expression of the ␣-subunit isoforms of Na - brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In ϩ osmoregulatory tissues, one isoform was expressed predominantly K -ATPase has been observed (20, 30, 52). Recently, it was in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney suggested that, in rainbow trout, a shift in salinity between by 10.220.33.6 on January 9, 2017 and intestine (FXYD12). Expression of several FXYD genes in fresh water (FW) and seawater (SW) is associated with a shift kidney and gill differed between fresh water and seawater salmon, in branchial expression of two of these isoforms (␣1a and ␣1b; suggesting significance during osmoregulatory adaptations. In addi- Ref. 49). FXYD proteins may well have regulatory influence tion to identifying novel FXYD isoforms, these studies are the first to on both cellular and transepithelial ion transport in teleosts, show the tissue dependence in their expression and modulation by similar to what is known for mammals. For example, during salinity in any teleosts. transitions between FW and SW, expression of FXYD proteins may contribute to the switch from hyper- to hypoosmoregula- Atlantic salmon; sodium-potassium-adenosinediphosphatase; osmo- ϩ ϩ regulation; Salmo salar; quantitative polymerase chain reaction tion by altering the kinetic behavior of Na -K -ATPase in branchial chloride cells and in epithelial cells of other osmo- regulatory tissues. In addition, due to the sensitivity of some DURING THE LAST DECADE, THERE has been an increasing focus on FXYD proteins to kinase signaling, they may also be involved in the role of FXYD proteins in regulation of cellular ion trans- altered tissue responsiveness to endocrine or paracrine signals. port. FXYD proteins constitute a family of small proteins with Despite the importance of FXYD proteins in determining ion a single transmembrane segment (for review, see Refs. 17, 26, transport characteristics in mammalian tissues, studies of these 28). They are named after the conserved extracellular motif, potential regulators in teleosts are, however, largely missing. The and all have a high degree of homology in a 35-amino acid propositions of the present study are as follows: 1) there may be stretch adjacent to and in the transmembrane domain, whereas more teleost FXYD isoforms than the three identified in zebrafish homology outside this area is low (55). In mammals, eight (55); 2) some isoforms may be expressed in a tissue-specific different FXYD isoforms have been identified, and association manner; and 3) salinity change may induce altered expression with the Naϩ-Kϩ-ATPase has been reported to affect the patterns in osmoregulatory tissues. The anadromous Atlantic ϩ ϩ enzyme affinity for substrate (Na ,K , and ATP) and/or Vmax salmon were used as the choice model to test these hypotheses. (e.g., FXYD1, Ref. 39; FXYD2, Ref. 4; FXYD3, Ref. 19; METHODS FXYD4, Ref. 6; FXYD5, Ref. 41; FXYD7, Ref. 7). It is well established that three of four mammalian isoforms of the Fish and sampling. One-year-old Atlantic salmon (Salmo salar, catalytic ␣-subunit of Naϩ-Kϩ-ATPase are expressed in a A¨ tran stock) were obtained in early March from The Danish Centre Address for reprint requests and other correspondence: C. K. Tipsmark, The costs of publication of this article were defrayed in part by the payment Institute of Biology, Univ. of Southern Denmark, Campusvej 55, DK-5230 of page charges. The article must therefore be hereby marked “advertisement” Odense M, Denmark (e-mail: [email protected].). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.ajpregu.org 0363-6119/08 $8.00 Copyright © 2008 the American Physiological Society R1367 R1368 EXPRESSION OF FXYD GENES IN A TELEOST for Wild Salmon (Randers, Denmark; http://www.vildlaks.dk/). They genes were chosen as a mammalian representative with FXYD1 were kept at a constant photoperiod (12:12-h light-dark) in recircu- (EDL23964), FXYD2 (EDL25650), FXYD3 (EDL23956), FXYD4 lated FW in indoor 500-liter tanks on the Odense Campus of the (EDK99588), FXYD5 (EDL23967), FXYD6 (AAH42579), and University of Southern Denmark until sampling in January, when the FXYD7 (EDL23965). Three chicken (Gallus gallus) representatives fish weight was 100–150 g. SW-acclimated fish were obtained by were found: a chicken protein similar to mouse FXYD2 (expect value transferring a batch of fish to 28 parts per thousand (ppt) artificial SW 6e-12; BX931806), a chicken protein weakly similar to mouse (Red Sea, Eliat, Israel) 2 wk before sampling. The fish were fed ad FXYD5 (expect value 1e-6; BU252825), and chicken FXYD6 libitum three times a week with pelleted trout feed. When sampled, (NP001074348). Amphibian representatives were found in the Afri- fish were stunned with a blow to the head, and blood was collected can clawed frog (Xenopus laevis): frog FXYD1 (NP001091365), frog with a heparinized syringe from the caudal vessels after which the fish FXYD2 (NP001081551), frog protein similar to mouse FXYD3 (ex- was killed. The first gill arch, posterior trunk kidney, anterior intes- pect value 9e-19; CX132847), a long frog FXYD5-like protein tine, heart, muscle, brain, and liver were dissected and, when needed, (BC085206), frog FXYD6 (NP001086408), and frog protein weakly trimmed free of cartilage, fat, and connective tissue and then instantly similar to mouse FXYD7 (expect value 7e-8; CF548198). A known frozen in liquid N2 until analysis. The experimental protocols were elasmobranch FXYD protein from spiny dogfish (Squalus Acanthias) approved by the Danish Animal Experiments Inspectorate, in accor- was included (FXYD10, AJ556170). A sea lamprey (Petromyzon dance with the European convention for the protection of vertebrate marinus) protein weakly similar to mouse FXYD1 (expect value Downloaded from animals used for experiments and other scientific purposes (no. 6e-10; CO551377) was used as the outgroup. 86/609/EØF). The predicted FXYD genes from Atlantic salmon and zebrafish Extraction of RNA and cDNA synthesis. Total RNA was extracted (Danio rerio), together with those of mouse and other vertebrates, by the TRIzol procedure (Invitrogen, Carlsbad, CA) using maximally were aligned using ClustalW. A majority rule consensus tree was 100 mg tissue/ml TRIzol, according to manufacturer’s recommenda- generated using SEQBOOT, PROML, and CONSENSE, all programs tions. Total RNA concentrations were determined by measuring ratio in the PHYLIP package (22). Maximum likelihood (ML) was used for of absorbance at 260 nm to that at 280 nm in duplicate with a the phylogenetic tree construction. A total of 1,000 bootstraps were http://ajpregu.physiology.org/ NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, used to test the consistency of the grouping within the tree. A Wilmington, DE). Purity (ratio of absorbance at 260 nm to that at 280 parsimonious model was used to further evaluate the main nodes found nm) ranged from 1.9 to 2.2. One-microgram RNA was treated with 1 with the ML method, also with the PHYLIP package (SEQBOOT, unit RQ1 DNase (Promega, Madison, WI) for 40 min at 37°C in a PROTPARS, and CONSENSE).