FXYD Proteins Research Articles

THU578 Concept: Role Of Spiral Steroids (SS) During Pregnancy

Abstract Disclosure: F.I. Chasalow: None. Background: Earlier, my laboratory reported 3 novel endogenous steroids (SS) that cross react with digoxin-specific antibodies (DLM). The SS are readily distinguished from all other steroids by 3 characteristics: [1] each is a phosphocholine diester, [2] each has more than 21 carbon atoms in the steroid fragment and [3] the extra carbons form an E-ring with a cyclic lactone. The discovery included a biosynthetic scheme with known enzymes and no need for a ‘magic’ step. Function: NaK-ATPase has 3 subunits, designated FXYD, α, and β. Affinity for Na+ and K+ ions is regulated by FXYD proteins. Each SS matches a tissue-specific α-subunit. Each α-subunit has 2 different binding sites for steroids. One site binds a SS; the ligand of the other site is unknown. For example, Ionotropin is specific for the α subunit present in cardiac and renal tissue. Kaleotropin is isolated from gonads, but the α subunit which matches each specific ovarian cell type is unknown. Galotropin (G-trop): One of the SS, G-trop, was detected as a DLM in human breast cyst fluids, specifically cysts with high K+ ion levels. Solubility properties distinguished it from previously known cardiac glycosides and, like other DLM, it inhibited NaK-ATPase. G-trop was also detected in milk from cows, sheep and goats. Ions derived from Ionotropin (m/z=341 Da) or Kaleotropin (m/z=369 Da) were not present in milk extracts. On LC-MS, G-trop fragmented to an ion at m/z= 381 Da. Trial and error analysis suggests C25O4H34. No other formula is consistent with m/z=381 Da. G-trop: G-trop wasn’t present in serum from children. At 22-26 weeks of gestation, whether or not a pregnant woman had pre-eclampsia, serum had low levels of G-trop. Maternal G-trop increases during the 3rd trimester, coincidentally with initiation of milk production; simultaneously, K+ ion levels in milk increased. Pre-eclampsia. In a pilot study, 65% of patients (n=20) with pre-eclampsia had elevated levels of Ionotropin precursor and 30% had elevated levels of Kaleotropin precursor. 25% of patients with pre-eclampsia develop life-threatening hypertension or seizures, typical of the expected response to elevated Ionotropin levels. Therapy includes immediate delivery of the infant at 30-36 weeks of gestation. Maternal G-trop synthesis has not yet begun and mother’s milk has low K+ ion levels. Elevated maternal Kaleotropin is not the cause of maternal hypertension, suggesting a specific gestational maternal α-subunit is its target. Kaleotropin from placenta may be the signal to respond to maternal hypokalemia. Necrotizing enterocolitis (NEC):In utero the fetus does not absorb K+ from the GI tract because fetal need for K+ ion is provided via the placenta. Concept: After premature delivery, without G-trop in milk, the infant can’t absorb K+ from the GI tract; the GI tract fails to grow and tight junctions do not form. As a result, infants are at high risk for developing NEC. Presentation Date: Thursday, June 15, 2023

A switch in FXYD proteins dysregulates NKA in dilated cardiomyopathy.

In the heart, Na+ homeostasis is established by the Na+/K+-ATPase (NKA). Because NKA is functionally linked to the Na+/Ca2+ exchanger, changes in NKA activity influence Ca2+ levels and cardiac contractility. It is well known that NKA in the heart is inhibited by FXYD1, a member of the FXYD protein family. However, the cardiac expression of other FXYDs has not been determined. In this study, western blot analysis revealed that FXYDs 5, 6 and 7 are also expressed in the human myocardium. In patients with dilated cardiomyopathy (DCM), FXYD1 expression was decreased and FXYD6 expression was increased. Furthermore, immunofluorescence staining showed that upregulation of FXYD6 in DCM patients occurs in the cardiomyocytes, which are the cells primarily responsible for cardiac contraction. FXYD6, like FXYD1 inhibits NKA. Thus, FXYD6 upregulation may be compensating for FXYD1 downregulation in order to maintain cardiac contractility. However, we observed that FXYD6 is expressed as dimers in the heart, but binds to NKA as monomers. Using fluorescence resonance energy transfer (FRET) and western blotting, we found that FXYD6 dimerization occurs due to oxidative crosslinking of cysteine residues (C9, C60 and C62). Additionally, mutation of FXYD6 cysteine residues decreased FXYD6 dimerization and increased binding with NKA, as measured by FRET. Together, these results suggest that in DCM, NKA is dysregulated by a switch from FXYD1 to FXYD6, which can become oxidized to form dimers, sequestering monomeric FXYD6 away from NKA. Decreased inhibition of NKA would lead to decreased Ca2+ levels and cardiac contractility, both of which are features of DCM.

FXYD3 Expression Predicts Poor Prognosis in Renal Cell Carcinoma with Immunosuppressive Tumor Microenvironment.

Simple SummaryFXYD3 belongs to the protein-coding gene family associated with Na+/K+-ATPase enzymes and chloride ion channels. Recently, the biological role of FXYD3 has been reported in multiple cancers. Nevertheless, the prognostic value of FXYD3 expression has been undiscovered in clear renal cell carcinoma (KIRC). In this study, we assessed the datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) dataset (GSE29609). We found the FXYD3 high KIRC patients had distinct clinical characteristics, including hypoxia and poor overall survival. Furthermore, the algorithms discovered that FXYD3 mRNA levels were associated with tumor purity, multiple types of the tumor infiltrating lymphocytes (TILs) and several genes related to T cell exhaustion. In conclusion, FXYD3 predicts a poor prognosis associated with hypoxia, pro-tumor TILs, and T cell exhaustion in KIRC.FXYD3 is a protein-coding gene, belonging to the FXYD protein family associated with Na+/K+-ATPase enzymes and chloride ion channels. Accumulating evidence suggests the biological role of FXYD3 in multiple cancers. However, the prognostic value of FXYD3 expression in clear renal cell carcinoma (KIRC) is unclear. Therefore, we evaluated the clinical data with tumor-infiltrating lymphocytes (TILs) and immunoinhibitory gene expression data using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) dataset (GSE29609). First, the FXYD3 high KIRC patients had distinct clinical characteristics, including age, sex, disease stage, histological grade, and hypoxia-related gene expressions. Next, FXYD3 gene expression was correlated with poor overall survival in both TCGA and GSE29609 cohorts. The ESTIMATE algorithm revealed that higher FXYD3 mRNA levels were associated with increased infiltration of immune cells and tumor purity. Moreover, the FXYD3 high KIRC tissue harbored increased TILs such as B cells, CD8+ T cells, and M1 macrophage, whereas NK cells and neutrophils were decreased. In addition, we showed FXYD3 was co-expressed with several immunoinhibitory genes related to T cell exhaustion such as LGALS9, CTLA4, BTLA, PDCD1, and LAG3. In conclusion, FXYD3 is an unfavorable prognostic biomarker associated with hypoxia, pro-tumor TILs, and T cell exhaustion.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Displacement of Native FXYD Protein From Na+/K+-ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity.

The seven mammalian FXYD proteins associate closely with α/β heterodimers of Na+/K+-ATPase. Most of them protect the β1 subunit against glutathionylation, an oxidative modification that destabilizes the heterodimer and inhibits Na+/K+-ATPase activity. A specific cysteine (Cys) residue of FXYD proteins is critical for such protection. One of the FXYD proteins, FXYD3, confers treatment resistance when overexpressed in cancer cells. We developed two FXYD3 peptide derivatives. FXYD3-pep CKCK retained the Cys residue that can undergo glutathionylation and that is critical for protecting the β1 subunit against glutathionylation. FXYD3-pep SKSK had all Cys residues mutated to Serine (Ser). The chemotherapeutic doxorubicin induces oxidative stress, and suppression of FXYD3 with siRNA in pancreatic- and breast cancer cells that strongly express FXYD3 increased doxorubicin-induced cytotoxicity. Exposing cells to FXYD3-pep SKSK decreased co-immunoprecipitation of FXYD3 with the α1 Na+/K+-ATPase subunit. FXYD3-pep SKSK reproduced the increase in doxorubicin-induced cytotoxicity seen after FXYD3 siRNA transfection in pancreatic- and breast cancer cells that overexpressed FXYD3, while FXYD3-pep CKCK boosted the native protein’s protection against doxorubicin. Neither peptide affected doxorubicin’s cytotoxicity on cells with no or low FXYD3 expression. Fluorescently labeled FXYD3-pep SKSK was detected in a perinuclear distribution in the cells overexpressing FXYD3, and plasmalemmal Na+/K+-ATPase turnover could not be implicated in the increased sensitivity to doxorubicin that FXYD3-pep SKSK caused. FXYD peptide derivatives allow rapid elimination or amplification of native FXYD protein function. Here, their effects implicate the Cys residue that is critical for countering β1 subunit glutathionylation in the augmentation of cytotoxicity with siRNA-induced downregulation of FXYD3.

FXYD6 dimerization in heart failure

In the heart, Na+/K+ ATPase (NKA) activity maintains Na+ and K+ gradients, and indirectly influences intracellular calcium levels and cardiac contractility. The cardiac NKA is inhibited by phospholemman (PLM), a member of the FXYD protein family. However, the possible cardiac expression and function of other FXYDs has not been determined. In this study, Western blot analysis revealed that FXYD6, an inhibitor of NKA, is also expressed in human myocardium. FXYD6 expression was increased in failing human hearts, and we observed a high molecular weight species consistent with a FXYD6-FXYD6 dimer.

Stoichiometry of the sodium pump-phospholemman regulatory complex

The sodium-potassium ATPase (NKA) is the ion motive ATP-dependent transporter that establishes sodium and potassium gradients across the cell membrane, facilitating many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1), a member of the FXYD protein family. PLM inhibits NKA by reducing pump’s apparent affinity of the pump for Na+, which is relieved by PLM phosphorylation. In this study, we used time-correlated single-photon counting, FRET-microscopy and molecular dynamics simulations to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex.

FXYD proteins and sodium pump regulatory mechanisms.

The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

FXYD protein isoforms differentially modulate human Na/K pump function.

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1β1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1β1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

Gene expression profiling of proximal and distal renal tubules in Atlantic salmon (Salmo salar) acclimated to fresh water and seawater.

Euryhaline teleost kidneys undergo a major functional switch from being filtratory in freshwater (FW) to being predominantly secretory in seawater (SW) conditions. The transition involves both vascular and tubular effects. There is consensus that the glomerular filtration rate is greatly reduced upon exposure to hyperosmotic conditions. Yet, regulation at the tubular level has only been examined sporadically in a few different species. This study aimed to obtain a broader understanding of transcriptional regulation in proximal versus distal tubular segments during osmotic transitions. Proximal and distal tubule cells were dissected separately by laser capture microdissection, RNA was extracted, and relative mRNA expression levels of >30 targets involved in solute and water transport were quantified by quantitative PCR in relation to segment type in fish acclimated to FW or SW. The gene categories were aquaporins, solute transporters, fxyd proteins, and tight junction proteins. aqp8bb1, aqp10b1, nhe3, sglt1, slc41a1, cnnm3, fxyd12a, cldn3b, cldn10b, cldn15a, and cldn12 were expressed at a higher level in proximal compared with distal tubules. aqp1aa, aqp1ab, nka-a1a, nka-a1b, nkcc1a, nkcc2, ncc, clc-k, slc26a6C, sglt2, fxyd2, cldn3a, and occln were expressed at a higher level in distal compared with proximal tubules. Expression of aqp1aa, aqp3a1, aqp10b1, ncc, nhe3, cftr, sglt1, slc41a1, fxyd12a, cldn3a, cldn3b, cldn3c, cldn10b, cldn10e, cldn28a, and cldn30c was higher in SW- than in FW-acclimated salmon, whereas the opposite was the case for aqp1ab, slc26a6C, and fxyd2. The data show distinct segmental distribution of transport genes and a significant regulation of tubular transcripts when kidney function is modulated during salinity transitions.

Positive correlation of gene expression between branchial FXYD proteins and Na+/K+-ATPase of euryhaline milkfish in response to hypoosmotic challenges

FXYD proteins are crucial regulators of Na+/K+-ATPase (NKA), which plays an important role in ion exchange by providing the driving force for other ion-transporting systems in the osmoregulatory organs, including the gills. In milkfish (Chanos chanos), gill NKA has been widely investigated and found to alter its expression (both mRNA and protein) and activity in response to environmental salinity changes. However, the expression and roles of the regulatory proteins of NKA, the FXYD proteins, in milkfish gills upon salinity challenge is not yet clear. Hence, 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 the effects of salinity on expression of gill fxyd and nka mRNA. Six milkfish FXYD proteins (CcFXYD) were identified. In milkfish gill, gill-specific Ccfxyd11 was the predominant member, followed by Ccfxyd9 and Ccfxyd8. Upon hypoosmotic challenges, increases in gill Ccfxyd11, Ccfxyd8, Ccnka α1, and Ccnka β1 mRNA as well as significantly positive correlations were observed. Moreover, after acute salinity changes, expression of gill Ccfxyd11 and Ccnka was found to change with ambient salinity, and significant positive correlations were also exhibited between Ccfxyd11 and Ccnka α1. Overall, these results revealed close relationships between CcFXYD11 and CcNKA α1 in milkfish gills, highlighting the potential roles of CcFXYD11 in osmoregulation.

FXYD Domain Containing Ion Transport Regulator 3 (FXYD3) is Over-Expressed in Germinal Centre Derived Aggressive Lymphomas and Plasma Cell Myeloma

The FXYD domain containing ion transport regulator 3 (FXYD3) belongs to the FXYD protein family, originally identified by Sweadner and Rael [1] basing on sequence similarity, and accounting in mammal for at least seven members.

Gene expression of Na+/K+-ATPase α-isoforms and FXYD proteins and potential modulatory mechanisms in euryhaline milkfish kidneys upon hypoosmotic challenges

Euryhaline milkfish (Chanos chanos) is an important economic species in Taiwan and is able to survive in both seawater (SW) and fresh water (FW). Upon salinity changes, euryhaline fish kidneys play a crucial role in osmoregulation. During this process, Na+/K + -ATPase (NKA) mainly functions in providing the driving force for the other ion-transporting systems. In the milkfish, NKA activities have been widely studied, yet the isoforms and modulatory mechanisms of the NKA α-subunits are unknown. Hence, this study was undertaken to reveal potential modulatory mechanisms, including the isoform composition of NKA α-subunits and FXYD proteins (the modulators of NKA), of NKA expression/activity in milkfish kidneys in response to salinity changes. Four isoforms (CcNKA α1a, α1c, α2, and α3) of NKA α-subunits with distinct tissue distribution were identified in milkfish. Among them, Ccnka α1a and Ccfxyd2 were predominantly expressed in the kidney. Upon salinity challenge, parallel expression patterns were found in renal Ccnka α1a and Ccfxyd2, showing an increase in the FW-acclimated milkfish but not in the SW-acclimated group. After transfer, no matter from SW to FW or FW to SW, most of the renal Ccnka and Ccfxyd genes (particularly Ccnka α1a and Ccfxyd2) significantly changed. Overall, these results suggest that the NKA activity of milkfish kidneys may be modulated via CcNKA α1a and CcFXYD2 for osmoregulation in response to salinity challenges.

Intestinal FXYD12 and sodium-potassium ATPase: A comparative study on two euryhaline medakas in response to salinity changes.

FXYD proteins are the regulators of sodium-potassium ATPase (Na+/K+-ATPase, NKA). In teleosts, NKA is a primary driving force for the operation of many ion transport systems in the osmoregulatory organs (e.g. intestines). Hence, the purpose of this study was to determine the expression of FXYD proteins and NKA α-subunit in the intestines of two closely related medakas (Oryzias dancena and O. latipes), which came from different salinity habitats and have diverse osmoregulatory capabilities, to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA was the most predominant in the intestines of both medakas. The association of FXYD12 and NKA in the intestines of the two medaka species was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, the localization of FXYD12 and NKA was similar in the intestines of the two medaka species. However, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels), as well as NKA activity differed between the medakas. These results showed that FXYD12 may play a role in modulating NKA activity in the intestines of the two medakas following salinity changes in the maintenance of internal homeostasis. These findings contributed to knowledge of the expression and potential role of vertebrate FXYD12, the regulators of NKA, upon salinity challenge.

FXYD8, a Novel Regulator of Renal Na+/K+-ATPase in the Euryhaline Teleost, Tetraodon nigroviridis.

FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates.

FXYD5 Protein Has a Pro-inflammatory Role in Epithelial Cells

The FXYD proteins are a family of small membrane proteins that share an invariant four amino acid signature motif F-X-Y-D and act as tissue-specific regulatory subunits of the Na,K-ATPase. FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing Vmax However, unlike other family members FXYD5 appears to have additional functions, which cannot be readily explained by modulation of transport kinetics. Knockdown of FXYD5 in MDA-MB-231 breast cancer cells largely decreases expression and secretion of the chemokine CCL2 (MCP-1). A related effect has also been observed in renal cell carcinoma cells. The current study aims to further characterize the relationship between the expression of FXYD5 and CCL2 secretion. We demonstrate that transfection of M1 epithelial cell line with FXYD5 largely increases lipopolysaccharide (LPS) stimulated CCL2 mRNA and secretion of the translated protein. We have completed a detailed analysis of the molecular events leading to the above response. Our key findings indicate that FXYD5 generates a late response by increasing the surface expression of the TNFα receptor, without affecting its total protein level, or mRNA transcription. LPS administration to mice demonstrates induced secretion of CCL2 and TNFα in FXYD5-expressing lung peripheral tissue, which suggests a possible role for FXYD5 in normal epithelia during inflammation.

FXYD11 mediated modulation of Na+/K+-ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments

FXYD proteins regulate Na+/K+-ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka (Oryzias dancena) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka-α was elevated 48h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka-α mRNA expression and NKA activity increased significantly 12h and 1h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein–protein interaction between branchial NKA and FXYD11 increased acutely 12h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1h following post-exposure to SW, without the elevation of protein–protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts.

Different Modulatory Mechanisms of Renal FXYD12 for Na(+)-K(+)-ATPase between Two Closely Related Medakas upon Salinity Challenge.

Upon salinity challenge, the Na+-K+-ATPase (NKA) of fish kidney plays a crucial role in maintaining ion and water balance. Moreover, the FXYD protein family was found to be a regulator of NKA. Our preliminary results revealed that fxyd12 was highly expressed in the kidneys of the two closely related euryhaline medaka species (Oryzias dancena and O. latipes) from different natural habitats (brackish water and fresh water). In this study, we investigated the expression and association of renal FXYD12 and NKA α-subunit as well as potential functions of FXYD12 in the two medakas. These findings illustrated and compared the regulatory roles of FXYD12 for NKA in kidneys of the two medakas in response to salinity changes. In this study, at the mRNA and/or protein level, the expression patterns were similar for renal FXYD12 and NKA in the two medakas. However, different patterns of NKA activities and different interaction levels between FXYD12 and NKA were found in the kidneys of these two medakas. The results revealed that different strategies were used in the kidneys of the two medaka species upon salinity challenge. On the other hand, gene knockdown experiments demonstrated that the function of O. dancena FXYD12 allowed maintenance of a high level of NKA activity. The results of the present study indicated that the kidneys of the examined euryhaline medakas originating from brackish water and fresh water exhibited different modulatory mechanisms through which renal FXYD12 enhanced NKA activity to maintain internal homeostasis. Our findings broadened the knowledge of expression and functions of FXYD proteins, the modulators of NKA, in vertebrates.

Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na(+)-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.