Academic literature on the topic 'FXYD proteins'

It is increasingly clear that alterations in Na+-K+-ATPase kinetics to fit the demands in specialized cell types is vital for the enzyme to execute its different physiological roles in diverse tissues. In addition to tissue-dependent expression of isoforms of the conventional subunits, α and β, auxiliary FXYD proteins appear to be essential regulatory components. The present study identified genes belonging to this family in Atlantic salmon by analysis of expressed sequence tags. Based on the conserved domain of these small membrane proteins, eight expressed FXYD isoforms were identified. Phylogenetic analysis suggests that six isoforms are homologues to the previously identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, while two additional isoforms were found (FXYD11 and FXYD12). Using quantitative PCR, tissue-dependent expression of the different isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, and liver. Two isoforms were expressed in several tissues (FXYD5 and FXYD9), while six isoforms were distributed in a discrete manner. In excitable tissues, two isoforms were highly expressed in brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In osmoregulatory tissues, one isoform was expressed predominantly in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney and intestine (FXYD12). Expression of several FXYD genes in kidney and gill differed between fresh water and seawater salmon, suggesting significance during osmoregulatory adaptations. In addition to identifying novel FXYD isoforms, these studies are the first to show the tissue dependence in their expression and modulation by salinity in any teleosts.

FXYD proteins belong to a family of small-membrane proteins. Recent experimental evidence suggests that at least five of the seven members of this family, FXYD1 (phospholemman), FXYD2 (γ-subunit of Na-K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), and FXYD7, are auxiliary subunits of Na-K-ATPase and regulate Na-K-ATPase activity in a tissue- and isoform-specific way. These results highlight the complexity of the regulation of Na+and K+handling by Na-K-ATPase, which is necessary to ensure appropriate tissue functions such as renal Na+reabsorption, muscle contractility, and neuronal excitability. Moreover, a mutation in FXYD2 has been linked to cases of human hypomagnesemia, indicating that perturbations in the regulation of Na-K-ATPase by FXYD proteins may be critically involved in pathophysiological states. A better understanding of this novel regulatory mechanism of Na-K-ATPase should help in learning more about its role in pathophysiological states. This review summarizes the present knowledge of the role of FXYD proteins in the modulation of Na-K-ATPase as well as of other proteins, their regulation, and their structure-function relationship.

Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH2O with NaCl for 48 h or adaptation to 700 mosmol/kgH2O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser68 (1–5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH2O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.

Four of the seven members of the FXYD protein family have been identified as specific regulators of Na,K-ATPase. In this study, we show that FXYD3, also known as Mat-8, is able to associate with and to modify the transport properties of Na,K-ATPase. In addition to this shared function, FXYD3 displays some uncommon characteristics. First, in contrast to other FXYD proteins, which were shown to be type I membrane proteins, FXYD3 may have a second transmembrane-like domain because of the presence of a noncleavable signal peptide. Second, FXYD3 can associate with Na,K- as well as H,K-ATPases when expressed in Xenopus oocytes. However, in situ (stomach), FXYD3 is associated only with Na,K-ATPase because its expression is restricted to mucous cells in which H,K-ATPase is absent. Coexpressed in Xenopus oocytes, FXYD3 modulates the glycosylation processing of the β subunit of X,K-ATPase dependent on the presence of the signal peptide. Finally, FXYD3 decreases both the apparent affinity for Na+ and K+ of Na,K-ATPase.

Proteins of the FXYD family act as tissue-specific regulators of the Na-K-ATPase. They are small hydrophobic type I proteins with a single-transmembrane span containing an extracellular invariant FXYD sequence. FXYD proteins are not an integral part of the Na-K-ATPase but function to modulate its catalytic properties by molecular interactions with specific Na-K-ATPase domains.

FXYD5 is a member of a family of tissue-specific regulators of the Na+-K+-ATPase expressed in kidney tubules. Previously, we have shown that FXYD5 interacts with the αβ-subunits of the Na+-K+-ATPase and increases its Vmax (Lubarski I, Pihakaski-Maunsbach K, Karlish SJ, Maunsbach AB, Garty H. J Biol Chem 280: 37717–37724, 2005). The current study further characterizes structural interaction and structure-function relationships of FXYD5. FXYD5/FXYD4 chimeras expressed in Xenopus laevis oocytes have been used to demonstrate that both the high-affinity association with the pump and the increase in Vmax are mediated by the transmembrane domain of FXYD5. Several amino acids that participate in the high-affinity interaction between FXYD5 and the α-subunit of the Na+-K+-ATPase have been identified. The data suggest that different FXYD proteins interact similarly with the Na+-K+-ATPase and their transmembrane domains play a key role in both the structural interactions and functional effects. Other experiments have identified at least one splice variant of FXYD5 with 10 additional amino acids at the COOH terminus, suggesting the possibility of other functional effects not mediated by the transmembrane domain. FXYD5 could be specifically bound to wheat germ agglutinin beads, indicating that it is glycosylated. However, unlike previous findings in metastatic cells, such glycosylation does not evoke a large increase in the size of the protein expressed in native epithelia and X. laevis oocytes.

The Na+-K+-ATPase is the primary electrogenic component driving transepithelial ion transport in the teleost gill; thus regulation of its level of activity is of critical importance for osmotic homeostasis. In the present study, we examined the dynamics of the gill-specific FXYD-11 protein, a putative regulatory subunit of the pump, in Atlantic salmon during seawater (SW) acclimation, smoltification, and treatment with cortisol, growth hormone, and prolactin. Dual-labeling immunohistochemistry showed that branchial FXYD-11 is localized in Na+-K+-ATPase immunoreactive cells, and coimmunoprecipitation experiments confirmed a direct association between FXYD-11 and the Na+-K+-ATPase α-subunit. Transfer of freshwater (FW)-acclimated salmon to SW induced a parallel increase in total α-subunit and FXYD-11 protein expression. A similar concurrent increase was seen during smoltification in FW. In FW fish, cortisol induced an increase in both α-subunit and FXYD-11 abundance, and growth hormone further stimulated FXYD-11 levels. In SW fish, prolactin induced a decrease in FXYD-11 and α-subunit protein levels. In vitro cortisol (18 h, 10 μg/ml) stimulated FXYD-11, but not FXYD-9, mRNA levels in gills from FW and SW salmon. The data show that Na+-K+-ATPase expressed in branchial mitochondrion-rich cells is accompanied by FXYD-11, and that regulation of the two proteins is highly coordinated. The demonstrated association of FXYD-11 and α-subunit strengthens our hypothesis that FXYD-11 has a role in modulating the pump's kinetic properties. The presence of putative phosphorylation sites on the intracellular domain of FXYD-11 suggests the possibility that this protein also may transmit external signals that regulate Na+-K+-ATPase activity.

Objective.To investigate the association of FXYD-3 expression with clinicopathological variables and PINCH in patients with ESCC.Patients and Methods.Expression of FXYD-3 protein was immunohistochemically examined in normal esophageal mucous (n=20) and ESCC (n=64).Results.Expression of FXYD-3 in the cytoplasm markedly increased from normal esophageal epithelial cells to primary ESCC (P=0.001). The expression of FXYD-3 was correlated with TNM stages and depth of tumor invasion. Furthermore, the cases with lymph node metastasis tended to show a higher frequency of positive expression than those without metastasis (P=0.086), and FXYD-3 expression tended to be positively related to the expression of PINCH (P=0.063). Moreover, the cases positive for both proteins had the highest frequency of lymph node metastasis (P=0.001). However, FXYD-3 expression was not correlated with patient’s gender (P=0.847), age (P=0.876), tumor location (P=0.279), size (P=0.7710.771), grade of differentiation (P=0.279), and survival (P=0.113).Conclusion.Overexpression of FXYD-3 in the cytoplasm may play an important role in the tumorigenesis and development in the human ESCC, particularly in combination with PINCH expression.

The Na +, K+-ATPase is an integral membrane protein that is essential for maintaining the ionic gradient in eukaryotes which is required for many important cellular processes including maintaining cell volume and the secondary transport of solutes. This project investigates the structure, function and physical properties of two classes of inhibitors of the Na+, K+-ATPase, FXYD proteins and the cardiac glycosides. This was achieved with a range of biophysical methods including solid-state NMR. The FXYD proteins are a family of seven physiological inhibitors of the Na+, K+-ATPase named after a shared PFXYD domain. They are transmembrane proteins of 64-178 amino acids which share sequence homology in their transmembrane domains but are sequentiaIIy diverse in their cytoplasmic domains. As the members of the FXYD family are tissue specifically expressed and they affect the activity of the Na +, K+ -ATPase in different ways it is thought that the variable cytoplasmic domains of these proteins are responsible for the unique functional features of each FXYD protein. This work focuses on two FXYD proteins: phospholemman (PLM) which has been linked to cardiac arrhythmia and Mat-S, a marker for several forms of cancer which has two isoforms varying in the lengths of their cytoplasmic domains (short form and long form). The initial hypothesis is that the cytoplasmic region ofPLM interacts with the Na+, K+-ATPase at one or more specific sites which causes a reduction of ATPase activity. This interaction is likely regulated by Ser 68 and/or Ser 63 phosphorylation and occurs in close proximity of the cell membrane surface due to the affinity of the cytoplasmic region of PLM for negatively charged lipid bilayers. This feature is unlikely shared with short form Mat-S as it does not have any identified phosphorylation sites but due to lack of data on long form Mat-S it is not clear whether or not its cytoplasmic region interacts with the Na+, K+-ATPase in a similar way to PLM. Initially, work was undertaken to develop a system to express and purify PLM and Mat-8. An existing method based on the expression of inclusion bodies was first investigated but was found to be inefficient. A new system was therefore developed which successfuIIy enabled the expression and purification of the short form of Mat-S. Further optimisation of this procedure will allow the expression and purification of isotopically labelled Mat-S suitable for analysis by NMR. Synthetic peptides corresponding to the cytoplasmic domains of PLM and Mat-S were studied in isolation of the transmembrane domains in order to investigate their interactions with Na+,K +-ATPase and with phospholipid membrane surfaces. The cytoplasmic domain of PLM has been found to interact with negatively charged phospholipid membrane surfaces and inhibit the Na+, K+-ATPase. These effects are reduced upon phosphorylation of Ser 68 suggesting phosphorylation has a role in regulating inhibition of the Na+, K+-ATPase by PLM. It was found here that the short isoform of Mat-S neither interacts with membrane surfaces nor inhibits Na+,K+-ATPase whereas the long isoform of Mat-S, which has an additional 26 amino acids in the cytoplasmic region, also binds to negatively charged cell membranes and inhibits the Na +, K+ -A'TPase albeit to a smaller extent. Motivated by these results, bioinformatic analysis identified a potential small linear Na", K+-ATPase binding motif; SxxRxS, which is present in PLM, long form Mat-S, several other binding partners of the Na +, K+ -A'TPase but not any other FXYD proteins. This motif in PLM was modeIIed onto a binding site predicted by Anchorsmap, showing both electrostatic and steric compatibility and favouring a site of interaction of the PLM cytoplasmic domain situated close to the membrane surface where interactions with lipid headgroups are possible. The cardiac glycosides are drugs used in heart failure and cardiac arrhythmia which are potent inhibitors of the Na+, K+-ATPase. Solid-state NMR methods were developed to investigate the position of cardiac glycosides in the high-affinity site within native membranes, by exploiting ouabain derivatised in the steroid moiety with a l3C-labeIIed acetonide bridge. By using the solid-state NMR technique of cross- polarisation, the natural abundance l3C nuclei from lipid headgroups and protein side chains can be observed together with a signature peak from the bound ouabain derivative. The paramagnetic broadening agent manganese was titrated into the membranes to probe the depth of the ouabain binding site. Paramagnetic broadening agents shorten the T2 times of local nuclei causing peak broadening with a distance-dependent relationship, thereby having greater effects on more accessible nuclei. The reduction of the ligand peak intensity was compared to the natural abundance l3C signals to estimate the depth of the acetonide bridge. The results suggest that the acetonide group attached to the steroid moiety is close to the surface of the protein. This methodology can now be used with double l3C-labelled ouabain to determine the orientation of the ligand in the binding site. The work presented here provides a range of insights into the structure function relationship of FXYD proteins and reveals a novel method for probing the depths of ligands within transmembrane proteins which can be applied to other systems.

Douleurs chroniques : Implication et potentiel thérapeutique des membres de la famille FXYDLes douleurs chroniques constituent un problème majeur de santé publique affectant près de 18% de la population mondiale. Elles ont des conséquences néfastes sur la qualité de vie des patients et engendrent des situations critiques sur le plan médical, sociologique et économique. Les traitements actuels sont relativement limités, souvent inefficaces et/ou présentent des effets secondaires délétères. De fait, une meilleure connaissance et une meilleure prise en charge de cette pathologie constituent des enjeux importants en recherche fondamentale et clinique.Dans ce contexte, mon projet de thèse a porté sur deux protéines, Fxyd2 et Fxyd7, qui appartiennent à la famille des protéines à motif FXYD qui contient 7 membres. Ces deux protéines sont décrites comme modulateurs de l’activité de la pompe Na,K-ATPase, et sont présentes dans des sous-types très spécifiques de neurones somatosensensoriels au sein des ganglions rachidiens dorsaux. La pompe Na,K-ATPase est décrite dans de nombreux phénomènes physiologiques et joue un rôle important dans l’excitabilité neuronale par le maintien du potentiel membranaire grâce à un jeu d'entrée et de sortie de sodium (Na+) et de potassium (K+). Le maintien de cet équilibre ionique est d’autant plus important que des phénomènes d’hyperexcitabilité neuronale sont souvent décrits dans le cadre des douleurs chroniques.Dans un premier objectif de mon projet de thèse, j’ai participé au développement d’une stratégie thérapeutique transposable à l’Homme basée sur une approche innovante de thérapie génique d’extinction. Ainsi, nous avons montré que l’usage d’oligonucléotides antisens lipidomodifiés dirigés contre le gène Fxyd2 et administrés par voie intrathécale permet un effet analgésique puissant chez des rats en condition de douleur neuropathique ou inflammatoire, aboutissant au retour à une sensibilité mécanique normale. De plus, des modifications chimiques conférant une meilleure stabilité de notre molécule thérapeutique permettent de prolonger son efficacité jusqu’à 10 jours.Mes travaux se sont portés, dans un deuxième objectif, sur la compréhension des modes d’action de Fxyd2 dans la physiopathologie des neurones somatosensoriels des ganglions rachidiens dorsaux notamment en identifiant ses partenaires protéiques par une approche protéomique. Ainsi, nous avons montré par spectrométrie de masse en tandem et par Proximity Ligation Assay, que Fxyd2 pouvait interagir de manière directe avec d’autres protéines que la sous unité ɑ1 de la pompe Na,K-ATPase en condition physiologique chez la souris. En effet, Fxyd2 semble interagir également avec la sous unité ɑ3 de cette pompe et aussi avec la PMCA, la GST et la Prdx6.Dans un troisième objectif, j’ai également étudié le rôle du gène Fxyd7 dans le système somatosensoriel aussi bien en condition normale que pathologique. Dans un premier temps, j’ai confirmé son profil d’expression au sein de sous populations neuronales de nocicepteurs peptidergiques, de mécanocepteurs et de neurones proprioceptifs dans les GRD de souris. Ensuite, en utilisant la souris Knock-Out pour le gène Fxyd7, j’ai procédé à des différents tests de motricité, d’équilibre et de sensibilité qui n’ont montré aucun défaut majeur chez ces souris en condition naïve. En condition de douleur neuropathique, avec le modèle SNL (Spinal Nerve Ligation), les tests de sensibilité mécanique ont montré aucune influence de la mutation, ni sur la phase aigüe de la douleur, ni sur sa chronicisation alors qu’en condition de douleur inflammatoire, avec le modèle CFA (Complete Freund’s Adjuvant), l’absence du gène empêche la chronicisation de la douleur inflammatoire de manière remarquable.Nos résultats montrent ainsi un potentiel thérapeutique majeur de deux membres de la famille FXYD pour traiter les douleurs chroniques
Chronic pain: Implication and therapeutic potential of FXYD protein members Chronic pain is a major public health problem affecting nearly 18% of the world’s population. It has deleterious consequences on patient’s quality of life and generates critical situations on the medical, sociological and economic levels. Current treatments are relatively limited, often ineffective and/or have deleterious side effects. In fact, better knowledge and an improved management of these pathologies is a major challenge for fundamental and clinical research.In this context, my thesis project is based on two different proteins, Fxyd2 and Fxyd7, which are members of a family of 7 proteins which contain a characteristic FXYD amino-acid motif. These two proteins have been described as modulators of the Na,K-ATPases’ activity, and are present in very specific somatosensory neurons of the dorsal root ganglia. The Na,K-ATPase pump is implicated in a large variety of physiological phenomena with a critical role in neuronal excitability by maintaining membrane potential thanks to the transfer of sodium (Na+) and potassium (K+). The maintenance of this ionic equilibrium is a crucial point since neuronal hyperexcitability has often been described in chronic pain.The first objective of my thesis was to develop a therapeutic strategy suitable for human therapy based on a very innovative gene extinction strategy. Thus, we showed that lipidomodified antisense oligonucleotides directed against the Fxyd2 gene and administered intrathecally induce a strong analgesic effect in neuropathic pain or in inflammatory pain models of rats, leading to normal mechanical sensitivity. Moreover, we showed that specific chemical modifications induce a better stability of our therapeutic molecule which prolongs its efficacy up to 10 days.In the second objective, my work was directed toward understanding the mechanisms of action of Fxyd2 in neuronal physiopathology in dorsal root ganglia, especially by identifying its protein partners using a proteomic approach. Thus, I showed by tandem mass spectrometry and by Proximity Ligation Assay that Fxyd2 could interact directly with proteins other than the ɑ1 subunit of the Na,K-ATPase in physiological conditions in mice. Indeed, Fxyd2 seems to interact also with the ɑ3 subunit of this pump and also with PMCA, GST and Prdx6.My third objective was to study the role of the Fxyd7 gene in the somatosensory system in normal and pathological conditions. In the first place, I used in situ hybridization to show its expression in specific neuronal subpopulations including peptidergic nociceptors, mechanoreceptors and in proprioceptive neurons in the mouse DRG. Then, using motor, equilibrium and mechanical sensitivity tests in Fxyd7 KO mice, I demonstrated the absence of major behavioral defects in these mice in normal conditions. In neuropathic pain conditions, using the SNL (Spinal Nerve Ligation) model, mechanical sensitivity tests did not reveal any influence of this mutation, neither in the acute nor chronic phases. However, in chronic inflammatory pain conditions induced by injection of CFA (Complete Freund’s Adjuvant), Fxyd7 null mutants failed to maintain pain responses. Thus Fxyd7 expression in DRG neurons appears to be specifically required for the maintenance of chronic inflammatory pain.Our results thus show a major therapeutic potential of two FXYD family members to treat chronic pain

博士
國立中興大學
生命科學系所
98
In the present study, three FXYD protein members were studied in pufferfish termed pFXYD. The goals of the research were to elucidate the molecular and functional mechanisms of osmoregulation. Subsequently, the salinity-dependent responses of pFXYD proteins revealed their functional role in the euryhaline teleost (Tetraodon nigroviridis) when faced with an osmoregulatory challenge. The Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound protein important for teleost osmoregulation. The enzyme is composed of two essential subunits; a catalytic α-subunit, and a glycosylated β-subunit responsible for membrane targeting of the enzyme. The smaller NKA γ-subunit, also known as FXYD2, is the first example of a small single transmembrane protein regulating NKA activity by interaction with the NKA α-subunit. Being the regulatory protein of NKA in mammals and elasmobranchs, it is intriguing to realize the expression and functions of FXYD protein in the euryhaline teleosts showing salinity-dependent changes of osmoregulatory organs NKA activity. The present study identified three cDNA sequences of pFXYDs confirmed by RT-PCR, including pFXYD5, pFXYD8, and pFXYD9. Amino acid sequences of pFXYD genes were deduced and the phylogenetical relationship of pFXYD proteins and other vertebrate FXYDs were analyzed. Pufferfish FXYD genes were expressed in the osmoregulatory organs of gill, kidney, and intestine of both freshwater (FW)- and seawater (SW)-acclimated euryhaline pufferfish. pFXYD9 showed differential expression across osmoregulatory tissues based on salinity acclimation. Based on real-time PCR, pFXYD9 was significantly higher in the gill and intestine of FW-acclimated pufferfish compared to SW-acclimated pufferfish. Conversely, renal mRNA expression of the pFXYD9 gene was higher in SW-acclimated fish when compared to FW-acclimated fish. On the other hand, pFXYD5 and pFXYD8 genes show a similar salinity acclimation dependent response in intestine only. Antibodies raised against partial amino acid sequences of the pFXYD8 and pFXYD9 proteins were detected in gill and kidney, however, in intestine, only pFXYD9 could be detected. On the other hand, an antibody raised against partial amino acid sequence of the pFXYD5 protein was applied in the immunoblots and a immunoreavtive band at 24 kDa was detected in three osmoregulatory organs. In branchial pFXYD5, branchial pFXYD9, renal pFXYD5, and intestine pFXYD9, the relative protein abundance was significantly higher in FW-acclimated group; but in branchial pFXYD8, renal pFXYD8, renal pFXYD9, and intestine pFXYD5 were significantly higher in SW-acclimated pufferfish. Function of pFXYD5, pFXYD8, and pFXYD9 all showed the ability to inhibit NKA activity by Xenopus oocyte experiments. Immunofluorescent staining of frozen sections provided direct evidence that branchial pFXYD9 and renal pFXYD8 were co-localized with NKA on MR cell. In addition, branchial pFXYD9 and renal pFXYD8 co-immunoprecipitated with NKA demonstrating their in vivo protein interaction. Comparisons of pFXYD mRNA and proteins expression with NKA activity were performed in gill, kidney, and intestine. These data provide evidence for the presences of pFXYD proteins, and their participation in osmoregulatory mechanisms by regulating NKA activity of the euryhaline teleost, Tetraodon nigroviridis.

博士
國立中興大學
生命科學系所
101
FXYD proteins are novel regulators of Na+-K+-ATPase (NKA). In fish subjected to salinity challenges, NKA in osmoregulatory organs (i.e., gills, kidneys, and intestines) is a primary driving force for many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on limited species. Based on their close phylogenetic relationships and diverse characteristics, the Oryzias species offer unique models for comparative and osmoregulatory studies. Among them, the brackish medaka (O. dancena) and the Japanese medaka (O. latipes) were model fish commonly used in experiments of different fields. The purposes of the present study were to use the brackish medaka, a saltwater fish model, for illustrating the potential roles of FXYD proteins and to investigate the diversity of teleostean FXYD expression profiles in these two closely related euryhaline medaka upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species (OdFXYD and OlFXYD). In the osmoregulayory organs, most fxyd genes expressed, and certain fxyd expression was salinity-dependent. Among the cloned genes, fxyd11 and fxyd12 was expressed mainly and abundantly in the gills, kidneys, and intestines, respectively. In gills of the brackish medaka, the OdFXYD11 protein interacted with the NKA α-subunit which was expressed at a higher level in freshwater-acclimated individuals relative to fish in the other salinity groups. Salinity changes led to different effects on the OdFXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. This finding (non-correlated expression patterns) is the first report of teleost FXYD proteins in a chronic (i.e., acclimated) rather than an acute (i.e., time-course) salinity challenge experiment. On the other hand, the function of OdFXYD12 was demonstrated to be able to maintain a high-level NKA activity. To our knowledge, this is the first study to illustrate the functions of teleost FXYD12 protein. Taken together, the present study inferred that the FXYD11 might play a crucial role in gills via increasing NKA activity in response to salinity challenge and revealed that FXYD12 was involved in osmoregulation/ionoregulation of internal osmoregulatory organs (i.e., kidneys and intestines) via enhancing NKA activity.

碩士
國立中興大學
生命科學系所
105
Milkfish (Chanos chanos) is an important economic species in Taiwan. Being a euryhaline teleost, milkfish is able to survive in both fresh water (FW) and seawater (SW). When euryhaline fish were exposed to salinity changes, their gills and kidneys play an important role in osmoregulation. The main function of the gill ionocyte and epithelial cells in nephrons of kidneys is ionoregulation and reabsorption of osmolytes, respectively. For osmolytes reabsorption, Na+, K+-ATPase (NKA; sodium potassium pump) mainly functions in providing driving force for the other secondary ion transporters. The NKA was consisted of α and β subunit. Three forms of NKA α isoforms in fish (α1-3) were found. In teleosts, paralogous genes of the α1 subunit of NKA named ‘‘nka α1a, nka α1b, and nka α1c’’ have been sequenced in several species. In addition, in recent years a novel protein family of NKA regulator, named FXYD, was also found. The FXYD proteins belong to a family of small membrane proteins that associate with and play a role as modulators of NKA. In milkfish, NKA activities have been widely studied, but lack evidences of NKA α1 paralogs and FXYD expressions, especially their expression in the kidney. Thus, this study was undertaken to clone and sequence the nka subunit isoforms from the kidney of milkfish, and to reveal the expression of renal NKA and FXYD proteins in euryhaline milkfish. Tissue distribution, adaptation of seawater (SW) or fresh water (FW) (long-term), and changes in renal NKA expression during FW- and SW- transfer (short-term) were performed. In this study, we found two NKA paralogs: nka α1b and nka α1c.The identities of amino acid sequences in the conserved region of NKA α1b and NKA α1c. Tissue-specific expression was found, in nka α1b, nka α2, fxyd2, fxyd7a, fxyd7b, fxyd8, fxyd9, and fxyd11. Among them, nka α1b and fxyd2 mainly expressed in the kidney; nka α2 have the highest expression in muscle; fxyd7 isoforms expressed in the brain; fxyd8 and fxyd9 showed small amounts of ubiquitous expression in various tissues, but most of them expressed in muscle and heart, respectively; fxyd11 only exists in the gill. In addition, nka α1c and nka α3 expressed ubiquitously in various tissues. The mRNA expression of nka α1b, nka α1c and fxyd2 in long-term experiments was significantly different in kidneys. They expressed higher in FW-acclimated milkfish. When milkfish was transferred from FW to SW for 12 hours and 1 days, nka α1b and fxyd2 expression decreased in the kidney, respectively. However, there was no significant change found in nka α1c when milkfish were transferred to SW (from FW) or FW (from SW) in short-term (one week). These results indicated that expression of NKA α1b, α1c, as well as FXYD2, were salinity-dependent in the long-term group. In the short-term experiment group, nka α1b and fxyd2 seemed to participate in ion absorptions, so after transfer from FW to SW they declined rapidly. Significant changes of fxyd8 and fxyd9 were only found in salinity transfer. On the other hand, nka α1c revealed no significant change. So nka α1c might function in long-term salinity acclimation in the milkfish.

碩士
國立中興大學
生命科學系所
104
Milkfish (Chanos chanos) is a euryhaline species and an important economic fish in Taiwan. In milkfish, branchial NKA have been widely studied, while the expression and potential roles of FXYD proteins were not clear yet. Therefore, this study illustrated the potential roles of milkfish branchial FXYD proteins in modulating NKA expression via identification and tissue distributions of FXYD proteins as well as effects of salinity on expression of gill fxyd and nka mRNA. This study identified five milkfish FXYD proteins (CcFXYD) via cDNA sequencing, sequence alignment, and phylogenetic tree analysis, including two CcFXYD7 isoforms, CcFXYD8, CcFXYD9, and CcFXYD11. Moreover, the identities of amino acid sequences in conserved region of FXYD proteins were highly similar among milkfish and the other teleosts (over 60%). In the tissue distribution, Ccfxyd8 and Ccfxyd9 expressed ubiquitously in various tissues of milkfish, while the two fxyd7 isoforms and fxyd11 expressed in a tissue-specific pattern. In the milkfish gill, the expression of Ccfxyd11 was the highest compared to that of the other Ccfxyds. After rearing in fresh water (FW) for at least one month, the mRNA levels of branchial Ccfxyd and nka were significantly higher in the milkfish rather than the seawater (SW) individuals. On the other hand, when milkfish was transferred to environments with different salinities (transfer from FW to SW or from SW to FW), the mRNA expression of branchial Ccfxyd8 and Ccfxyd9 changed slightly. Different mRNA levels of branchial Ccfxyd11and nka β1 were also found at all time-points since 48 hours post-transfer. In addition, the mRNA expression of branchial nka α1 decreased significantly at all time-points after transfer for 96-hour from FW to SW. Taken together, expressions of branchial Ccfxyd and nka genes of milkfish were salinity-dependent in both long-term and time-course experiments. These results showed that branchial CcFXYD proteins may be involved in gill osmoregulatory process via modulating NKA expression like the mammalian FXYD proteins. Parallel expression patterns of Ccfxyd and nka β1 implied that branchial CcFXYD played a role in positively modulation of NKA expression. Moreover, among the three branchial Ccfxyd, expression of Ccfxyd11 quickly reached stable levels after salinity challenge, showing that Ccfxyd11 may be able to efficienty assist milkfish in acclimation to environemenal salinity changes. The osmoregulatory roles of branchial CcFXYD8 and CcFXYD9, however, need to be clarified in future.