Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-subunit in response to stress

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. Na-K-ATPase; cellular stress; regulation     

Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change

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, alpha and beta, 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.     

FXYD2c Plays a Potential Role in Modulating Na+/K+-ATPase Activity in HK-2 Cells Upon Hypertonic Challenge

Na⁺/K⁺-ATPase (NKA) is a widely found and important transporter in mammals. The kidney is a major osmoregulatory organ of which the proximal tubules play a crucial role in the maintenance of ionic homeostasis functioning via salt and water reabsorption. FXYD (FXYD domain-containing protein) 2, the γ-subunit of NKA, is the first identified and the most abundant member of FXYD family, affecting the sodium/potassium affinity of NKA in the kidney. Based on DNA microarray analysis, the expression levels of fxyd2 gene are markedly increased upon hypertonic challenge. Combined with bioinformatic analysis using the NCBI database, we identified an unnamed protein with 145 amino acids, of which the N-terminus involved the FXYD sequence similar to FXYD2a and FXYD2b, and thus, named as FXYD2c. However, the role of FXYD2c protein in the regulation of NKA expression in the kidney has not been elucidated. In this study, we found that the mRNA and protein levels of FXYD2c were significantly increased upon hypertonic challenge. Immunoprecipitation data revealed that FXYD2c interacts with the NKA α1 subunit. Subsequently, the functional inhibition of fxyd2c using short hairpin RNA abrogated NKA activity. Taken together, our study offers novel insight into the potential function of FXYD2c in promoting NKA activity upon hypertonic challenge in HK-2 cells.     

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

FXYD proteins are novel regulators of Na⁺-K⁺-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the 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 only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.     

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.     

A study of the membrane association and regulatory effect of the phospholemman cytoplasmic domain

Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.     

CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the gamma-subunit of the Na,K-ATPase (NKA) that modulates the Na,K-pump function in the kidney. Here, we report that, similarly to gamma(a) and gamma(b) splice variants, the FXYD protein CHIF (corticosteroid-induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K-pump transport when expressed in Xenopus oocytes. In contrast to gamma(a) and gamma(b), which both decrease the apparent Na+ affinity of the Na,K-pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable gamma-subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the gamma-subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue-specific regulator of NKA which probably plays a crucial role in aldosterone-responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.     

FXYD proteins: novel regulators of Na,K-ATPase

Members of the FXYD protein family are small membrane proteins which are characterized by an FXYD motif, two conserved glycines and a serine residue. FXYD proteins show a tissue-specific distribution. Recent evidence suggests that 6 out of 7 FXYD proteins, FXYD1 (phospholemman), FXYD2 (gamma subunit of Na,K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (Ric) and FXYD7 associate with Na,K-ATPase and modulate its transport properties e.g. its Na+ and/or its K+ affinity in a distinct way. These results highlight the complex regulation of Na+ and K+ transport which is necessary to ensure proper tissue functions such as renal Na+-reabsorption, muscle contractility and neuronal excitability. Moreover, mutation of a conserved glycine residue into an arginine residue in FXYD2 has been linked to cases of human hypomagnesemia indicating that dysregulation of Na,K-ATPase by FXYD proteins may be implicated in pathophysiological states. A better characterization of this novel regulatory mechanism of Na,K-ATPase may help to better understand its role in physiological and pathophysiological conditions.     

TRIM family proteins: roles in proteostasis and neurodegenerative diseases

Neurodegenerative diseases (NDs) are a diverse group of disorders characterized by the progressive degeneration of the structure and function of the central or peripheral nervous systems. One of the major features of NDs, such as Alzheimer''s disease (AD), Parkinson''s disease (PD) and Huntington''s disease (HD), is the aggregation of specific misfolded proteins, which induces cellular dysfunction, neuronal death, loss of synaptic connections and eventually brain damage. By far, a great amount of evidence has suggested that TRIM family proteins play crucial roles in the turnover of normal regulatory and misfolded proteins. To maintain cellular protein quality control, cells rely on two major classes of proteostasis: molecular chaperones and the degradative systems, the latter includes the ubiquitin-proteasome system (UPS) and autophagy; and their dysfunction has been established to result in various physiological disorders including NDs. Emerging evidence has shown that TRIM proteins are key players in facilitating the clearance of misfolded protein aggregates associated with neurodegenerative disorders. Understanding the different pathways these TRIM proteins employ during episodes of neurodegenerative disorder represents a promising therapeutic target. In this review, we elucidated and summarized the diverse roles with underlying mechanisms of members of the TRIM family proteins in NDs.     

Splice Variants of the Gamma Subunit (FXYD2) and Their Significance in Regulation of the Na,K-ATPase in Kidney

The recent discovery of a family of single-span membrane proteins (the FXYD proteins) introduced a new direction to the rather complicated area of regulation of Na, K-ATPase. At least six members of the family have been shown to associate with the Na, K-ATPase in a cell- and tissue-specific manner, while four of them, namely the γ subunit (FXYD2), CHIF (FXYD4), phospholemman (FXYD1), and dysadherin (FXYD5) have been identified in kidney. All four exhibited different effects on the properties of the pump in heterologous expression systems. Taken along with their non-overlapping expression patterns in the nephron, this provides a potential structural basis for the segment-specific properties of the Na, K-ATPase that had been reported in a number of papers on kidney physiology. This brief review summarizes our own contributions on structure/functional characterization of one of the family members, the γ subunit (FXYD2). The focus is on splice variants of γ, their structural similarity and yet distinct effects conferred to Na, K-ATPase.     

The changing landscape of protein allostery

It is becoming increasingly clear that the fundamental capacity to undergo conformational change in response to ligand binding is intrinsic to proteins. This property confers on proteins the ability to be allosterically modulated in order to shift substrate binding affinities, alter enzymatic activity or regulate protein-protein interaction. How this allosteric modulation occurs--the pathways of communication, the shifting of conformational ensembles and the altered molecular dynamics--has received considerable attention during the past two years. Recent progress has helped outline the molecular origins of allostery in proteins as diverse as Hsp70 molecular chaperones and signal integrating proteins, such as WASP. In addition, allosteric properties have been successfully engineered into proteins for drug design or the development of novel biosensors. Methodological advances have provided exciting prospects for new insights and new biological roles of allosteric systems have been uncovered.     

FXYD7, mapping of functional sites involved in endoplasmic reticulum export, association with and regulation of Na,K-ATPase

The brain-specific FXYD7 is a member of the recently defined FXYD family that associates with the alpha1-beta1 Na,K-ATPase isozyme and induces an about 2-fold decrease in its apparent K+ affinity. By using the Xenopus oocyte as an expression system, we have investigated the role of conserved and FXYD7-specific amino acids in the cellular routing of FXYD7 and in its association with and regulation of Na,K-ATPase. In contrast to FXYD2 and FXYD4, the studies on FXYD7 show that the conserved FXYD motif in the extracytoplasmic domain is not involved in the efficient association of FXYD7 with Na,K-ATPase. On the other hand, the conserved Gly40 and Gly29, located on the same face of the transmembrane helix, were found to be implicated both in the association with and the regulation of Na,K-ATPase. Mutational analysis of FXYD7-specific regions revealed the presence of an ER export signal at the end of the cytoplasmic tail. Deletion of a C-terminal valine residue in FXYD7 significantly delayed and decreased its O-glycosylation processing and retarded the rate of its cell surface expression. This result indicates that the C-terminal valine residue is involved in the rapid and selective ER export of FXYD7, which could explain the observed post-translational association of FXYD7 with Na,K-ATPase. In conclusion, our study on FXYD7 provides new information on structural determinants of general importance for FXYD protein action. Moreover, FXYD7 is identified as a new member of proteins with a regulated ER export, which suggests that, among FXYD proteins, FXYD7 has a particular regulatory function in brain.     

Congenital myasthenic syndromes: A diverse array of molecular targets

The neuromuscular junction (NMJ) has served as a prototype for understanding mechanisms underlying synaptic transmission over the past 50 years. More recently, analysis of congenital myasthenic syndromes (CMS) revealed a diverse array of molecular targets and delineated their contributions to synaptic function. Clinical, electrophysiologic and morphologic studies have paved the way for detecting CMS-related mutations in proteins such as choline acetyltransferase acetylcholinesterase, the acetylcholine receptor, rapsyn, and the voltage-gated sodium channel of the Na_v1.4 type. Further studies of the mutant proteins have allowed us to correlate the effects of the mutations with predicted alterations in protein structure. In this review, we focus on the symptomatology of the CMS, consider the factors that impair neuromuscular transmission, survey the mutations that have been uncovered in the different synaptic proteins, and consider the functional implications of the identified mutations.     

The novel functions of ubiquitination in signaling

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.     

FXYD2, a γ subunit of Na+,K+-ATPase,maintains persistent mechanical allodynia induced by inflammation

Na⁺,K⁺-ATPase (NKA) is required to generate the resting membrane potential in neurons. Nociceptive afferent neurons express not only the α and β subunits of NKA but also the γ subunit FXYD2. However, the neural function of FXYD2 is unknown. The present study shows that FXYD2 in nociceptive neurons is necessary for maintaining the mechanical allodynia induced by peripheral inflammation. FXYD2 interacted with α1NKA and negatively regulated the NKA activity, depolarizing the membrane potential of nociceptive neurons. Mechanical allodynia initiated in FXYD2-deficient mice was abolished 4 days after inflammation, whereas it persisted for at least 3 weeks in wild-type mice. Importantly, the FXYD2/α1NKA interaction gradually increased after inflammation and peaked on day 4 post inflammation, resulting in reduction of NKA activity, depolarization of neuron membrane and facilitation of excitatory afferent neurotransmission. Thus, the increased FXYD2 activity may be a fundamental mechanism underlying the persistent hypersensitivity to pain induced by inflammation.     

Interaction of FXYD10 (PLMS) with Na,K-ATPase from shark rectal glands. Close proximity of Cys74 of FXYD10 to Cys254 in the a domain of the alpha-subunit revealed by intermolecular thiol cross-linking

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.     

Structure-function relationships in heme-proteins

Biological systems rely on heme-proteins to carry out a number of basic functions essential for their survival. Hemes, or iron-porphyrin complexes, are the versatile and ubiquitous active centers of these proteins. In the past decade, discovery of new heme-proteins, together with functional and structural research, provided a wealth of information on these diverse and biologically important molecules. Structure determination work has shown that nature has used a variety of different scaffolds and architectures to bind heme and modulate functions such as redox properties. Structural data have also provided insights into the heme-linked protein conformational changes required in many regulatory heme-proteins. Remarkable efforts have been made towards the understanding of factors governing redox potentials. Site-directed mutagenesis studies and theoretical calculations on heme environments investigated the roles of hydrophobic and electrostatic residues, and analyzed the effect of heme solvent accessibility. This review focuses on the structure-function relationships underlying the association of heme in signaling and iron metabolism proteins. In addition, an account is given about molecular features affecting heme's redox properties; this briefly revisits previous conclusions in the light of some more recent reports.