FXYD proteins: new regulators of Na-K-ATPase
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《美国生理学杂志》
Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland
ABSTRACT
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 (-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.
ion transport regulation; protein-protein interaction; structure-function relationship
THE NA-K-ATPASE, OR NA/K PUMP, belongs to the protein family of P-type ATPases that are characterized by the formation of a phosphorylated intermediate during the catalytic cycle. By using the energy of the hydrolysis of ATP, the Na-K-ATPase, located in the plasma membrane, transports three Na + ions out of the cell in exchange for two K + ions into the cell ( 43). The Na-K-ATPase is an oligomeric protein. The catalytic -subunit with 10 transmembrane segments transports the cations, hydrolyzes ATP, and is the pharmacological receptor for cardiac glycosides. A type II, glycosylated -subunit functions as a molecular chaperone necessary for the correct membrane insertion of the -subunit during its synthesis and modulates its transport properties ( 38). Four - and three -isoforms have been identified, which show a tissue-specific expression and can potentially form 12 different Na-K-ATPase isozymes with distinct transport and pharmacological properties ( 9, 21).
The Na-K-ATPase is the transport system responsible for the maintenance of the Na + and K + gradients across the plasma membrane. These gradients are essential to maintain cell volume and membrane potential, and the Na + gradients provide the energy for the activity for secondary Na +-dependent transport systems that supply the cell with nutrients and regulate intracellular pH and Ca 2+ concentrations. The Na-K-ATPase is present in all cells to ensure basic cellular homeostasis but it also contributes to specialized tissue functions. In muscle cells, the activity of Na-K-ATPase is tightly coupled to the activity of a Na/Ca exchanger and thus favors contractility of heart and skeletal muscles. The presence of Na-K-ATPase in nerve and glial cells permits restoration of the Na + and K + gradients during neuronal activity and thus ensures neuronal excitability. Moreover, in the kidney Na-K-ATPase is exclusively located in the basolateral membrane of epithelial cells and thus becomes the driving force for Na + reabsorption essential to maintain extracellular volume and blood pressure.
In view of its important physiological role, it may be expected that dysfunction or dysregulation of the Na-K- ATPase may have severe pathophysiological consequences. However, only recently have mutations in Na-K-ATPase been associated with genetic diseases. Four different mutations in a single allele of the 2-isoform of the Na-K-ATPase, which cause loss of function, are linked to familial hemiplegic migraine type 2 (FHM2), a hereditary form of migraine characterized by aura and some hemiparesis ( 15, 26, 79, 95). Moreover, missense mutations in the gene for the 3-isoform of the Na-K-ATPase have been identified as a cause of rapid-onset dystonia-Parkinsonism (RDP; DYT12) ( 25). Dysregulation of Na-K-ATPase through a defective production or function of a tissue-specific regulator has been correlated with various disorders, including cardiovascular, neurological, renal, and metabolic diseases, but the direct link between altered pump function and defective regulation remains in most cases obscure ( 50, 76).
Numerous mechanisms are involved in the regulation of the Na-K-ATPase to adapt its activity and/or expression to changing physiological demands, permitting proper "housekeeping" and specialized tissue functions of Na-K-ATPase. The requirements for Na-K-ATPase modulation are likely to be greatest in response to physiological stimuli such as nerve impulse propagation, exercise, and changes in dietary Na + and K +.
Because intracellular Na + is the limiting factor for Na-K-ATPase activity, any change in the intracellular Na + concentration affects its transport rate. Moreover, peptide hormones or neurotransmitters may provoke phosphorylation of Na-K- ATPase by protein kinases, which modulates its cell surface expression ( 88). Finally, steroid hormones such as aldosterone affect and gene transcription, leading to an increased number of Na-K-ATPase pump units ( 30).
In addition to these regulatory effects mediated by hormones and neurotransmitters, recent experimental evidence has revealed a novel regulatory mechanism that involves interaction of the Na-K-ATPase with small-membrane proteins of the FXYD family. In contrast to hormonal regulation, interaction of FXYD proteins does not produce a change in Na-K-ATPase expression but rather modifies the transport properties of the Na-K-ATPase in a tissue- and isoform-specific way.
THE FXYD PROTEIN FAMILY
Sweadner and Rael ( 86) have defined the gene family of FXYD proteins based on the invariant amino acids in a signature sequence containing the FXYD motif and two conserved glycine and a serine residue. In mammals, this family contains seven members including FXYD1 (or phospholemman) ( 71); FXYD2 (or the -subunit of Na-K-ATPase) ( 33, 59); FXYD3 (or mammary tumor marker Mat-8) ( 67); FXYD4 (or corticosteroid hormone-induced factor CHIF) ( 5); FXYD5 (or related to ion channel RIC or dysadherin) ( 34); FXYD6 (or phosphohippolin) ( 100); and FXYD7 ( 7) ( Fig. 1). Moreover, FXYD2 from Xenopus laevis ( 8) and a phospholemman-like protein from shark ( 55) have been cloned. FXYD proteins contain 61–95 amino acids, with the exception of FXYD5, which has 178 amino acids due to an NH 2-terminal extension. FXYD1 ( 71), FXYD2 ( 8), FXYD4 ( 6), and FXYD7 ( 7) are type I membrane proteins with a single membrane span and the COOH terminus exposed to the cytosol. FXYD1 and FXYD4 achieve this membrane orientation after cleavage of a signal peptide, whereas FXYD2 and FXYD7 have no signal peptide. Circular dichroism and NMR spectra predict that the transmembrane domains (TMs) of FXYD1, 2, 3, and 4 adopt an -helical conformation ( 24, 89). For years after their cloning, the functional role of FXYD proteins remained unknown. Because FXYD1 ( 63), FXYD3 ( 67), FXYD4 ( 5), and FXYD5 ( 34) induce ion-specific conductances when overexpressed in X. laevis, it was long thought that FXYD proteins may be ion channels or regulators of ion channels. However, the physiological significance of these observations remains unclear because induction of ion conductances was often observed only under nonphysiological conditions. Experimental evidence then showed that FXYD2, known for over 20 years to be associated with renal Na-K-ATPase ( 33), indeed modulates Na-K-ATPase activity ( 8). This promoted intense research that showed, until now, that at least five of the seven FXYD proteins interact with Na-K-ATPase and regulate its function. Thus, even though it is not known whether Na-K-ATPase regulation is the only function of FXYD proteins, it is likely that all FXYD proteins are tissue-specific auxiliary subunits of Na-K-ATPase. The experimental criteria that have been used to support this hypothesis are 1) coimmunoprecipitation of FXYD proteins with Na-K-ATPase both in expression systems and in tissues, 2) distinct modulation of Na-K-ATPase activity by different FXYD proteins, 3) Na-K-ATPase isozyme-specific association with and/or modulation by different FXYD proteins, and 4) FXYD protein-deficient mice. Below is a summary of the most recent studies that support that FXYD proteins are auxiliary subunits of Na-K-ATPase and deals with the characterization of each FXYD protein (some relevant structural and functional properties of FXYD proteins are summarized in Table 1). (For further reference, see Refs. 18, 20, 92.)
FXYD1 (PHOSPHOLEMMAN)
FXYD1 is mainly expressed in the heart, skeletal muscle, and liver ( 10, 71). Detection of FXYD1 in the juxtaglomerular apparatus of the kidney indicates that FXYD1 can also be expressed in particular regions of other tissues ( 99). Expression of FXYD1 in X. laevis oocytes ( 63) or addition of FXYD1 to planar bilayers ( 17, 62) induces a Cl –-selective conductance that is activated by hyperpolarizing voltages. Moreover, FXYD1 selectively transports the zwitterionic amino acid taurine ( 62), an osmolyte of animal cells. In response to cell swelling, taurine efflux and the regulatory decrease in cell volume increase in FXYD1-transfected cells ( 64). Finally, taurine efflux in astrocytes is inhibited after a decrease in endogenous FXYD1 by antisense oligonucleotides ( 65). Thus, based on these observations, it is believed that FXYD1 plays a specific role in muscle contractility and cell volume regulation.
The recent experimental evidence that FXYD1 interacts with Na-K-ATPase adds a new perspective to the potential physiological role of FXYD1. FXYD1 interacts specifically with 1-isozymes in native cardiac and skeletal muscle ( 19, 35, 85) and with 1, 2, and 3 isoforms in the cerebellum and choroid plexus ( 32). After expression in X. laevis oocytes, FXYD1 decreases the apparent Na + affinity and, to a lesser extent, the apparent K + affinity of Na-K-ATPase ( 19). In contractile tissues, the existence of low-Na +-affinity Na/K pumps may be necessary to permit efficient extrusion of increased intracellular Na + during action potentials and thus to control appropriate muscle contractility. Alternatively, the existence of a low-Na +-affinity Na/K pump associated with FXYD1 could lead to increased intracellular Na + concentrations, an increase in intracellular Ca 2+ concentrations due to inhibition of Ca 2+ efflux via the Na/Ca exchanger, and ultimately an increased contractility of heart and skeletal muscles. However, this hypothesis is not supported by recent data, which show that at high extracellular Ca 2+ concentrations, FXYD1 overexpression in rat myocytes leads to a reduction ( 102) and downregulation of FXYD1 and thus to increased ( 60) contractility. Overexpression of FXYD1 was correlated with inhibition of the Na/Ca exchanger of both its forward and reverse exchange activity ( 102), and downregulation with an enhancement of Na/Ca exchanger function ( 60). FXYD1 colocalizes with the Na/Ca exchanger, and coimmunoprecipitation experiments suggest a direct interaction between the two proteins ( 60), indicating that FXYD1 associates not only with Na-K-ATPase but also with the Na/Ca exchanger.
The physiological role of FXYD1 has been investigated in generations of FXYD1-deficient mice ( 45). These mice show an increased ejection fraction, an increased cardiac mass in the absence of hypertension, and a significant reduction in intrinsic Na-K-ATPase activity with little decrease in total Na-K- ATPase expression. These results were interpreted to indicate that FXYD1 modulates the Na-K-ATPase and that abolishment of FXYD1 expression reduces Na-K-ATPase activity, which leads to compensatory responses. However, because the Na-K-ATPase, Na/Ca exchanger, and FXYD1 are likely to colocalize in the transverse tubules of cardiomyocytes ( 61) and Na-K-ATPase 1- and 2-isozymes interact with the Na/Ca exchanger ( 28), the relative importance of the Na-K-ATPase or the Na/Ca exchanger, or perhaps other interacting proteins, in the functional effects in FXYD1-deficient mice remains to be elucidated. Moreover, the mechanism by which FXYD1 influences Na-K-ATPase in situ in native tissues is still unclear. The results obtained with FXYD1-deficient mice ( 45) suggest that FXYD1 stimulates rather than inhibits Na-K-ATPase, and no difference was seen in the apparent Na + affinity of the Na-K-ATPase in wild-type and FXYD1-deficient mice in contrast to the previously described decrease in the Na + affinity of Na-K-ATPase induced by mammalian FXYD1 ( 19) or by the phospholemman-like shark protein ( 55).
Phosphorylation may be at least one factor that regulates the putative multiple functions of FXYD1. Considered as the major substrate for protein kinase A in the heart ( 71), FXYD1 is phosphorylated on specific serine residues by PKA and PKC and the protein kinase never in mitosis A (NIMA) ( 53, 69, 96). PKA but not PKC phosphorylation of FXYD1 increases the FXYD1-mediated currents and its cell surface expression, whereas similar effects by NIMA kinases are independent of FXYD1 phosphorylation ( 69). On the other hand, phosphorylation by myotonic dystrophy kinase induces degradation of FXYD1 ( 68). Moreover, it has been suggested that PKC phosphorylation of a phospholemman-like protein from shark induces its dissociation from Na-K-ATPase and an increase in the Na + affinity of Na-K-ATPase ( 54). In contrast, in isolated sarcolemma, ischemia produces a substantial activation of Na-K-ATPase that was correlated with PKA phosphorylation of FXYD1 without influencing the association efficiency of FXYD1 with Na-K-ATPase ( 35). Finally, PKA phosphorylation of serine 68 in FXYD1 was implicated in the increased Na-K-ATPase pump currents measured in forskolin-treated ventricular myocytes ( 85).
In conclusion, compelling evidence suggests that FXYD1 is an auxiliary subunit of Na-K-ATPase in contractile tissues and in the brain. So far, the intrinsic physiological role in these tissues is not known. In the heart and skeletal muscle, Na-K-ATPase is tightly linked to contractility, and regulation of its kinetic properties by FXYD1 may have distinct effects on this tissue function. Because both 1- and 2-isozymes are implicated in cardiac contractility ( 28), the specific association of FXYD1 with 1-isozymes suggests a subtle difference in the mechanism underlying the function of 1-isozymes. The apparently discrepant results obtained regarding the mechanism of action of the regulatory effect of FXYD1 on Na-K-ATPase activity may partly be due to variations in its phosphorylation but could also be a result of an interplay between different functional roles of FXYD1, e.g., interaction with Na/Ca exchanger and channel formation.
FXYD2 (-SUBUNIT OF Na-K-ATPase)
The FXYD2 gene is located on chromosome 11q23 ( 87). It encodes two splice variants, FXYD2a and FXYD2b (a and b), which have been identified by mass spectroscopy and differ only in their most NH 2-terminal amino acids ( 49). FXYD2 is predominantly expressed in the kidney ( 59, 90) with segment specific distribution of FXYD2a and FXYD2b ( 4, 73) ( Table 2). FXYD2a and FXYD2b colocalize with Na-K- ATPase in the basolateral membrane of renal epithelial cells. No FXYD2 was detected in the collecting duct ( 4, 73).
FXYD2 was the first FXYD protein that was found to be associated with Na-K-ATPase ( 33, 59) and to produce a functional effect on its transport properties ( 8, 90). In contrast to FXYD1, no other functional role than that of a Na-K-ATPase regulator has been proposed, although it was reported that FXYD2 expressed in X. laevis oocytes activates Ca 2+- and voltage-gated, nonselective pores endogenous to the oocyte ( 80). Evidence provided from different experimental approaches suggests that FXYD2 may have several concomitant and independent effects on Na-K-ATPase function. FXYD2 was shown to increase the apparent K + affinity of Na-K-ATPase at high negative membrane potentials in both the presence and absence of extracellular Na + ( 6). On the other hand, FXYD2 decreases the apparent K + affinity at less negative membrane potentials but only in the presence of extracellular Na +, suggesting a shift in E1-E2 equilibrium toward the E1 conformation ( 6). FXYD2 also increases the affinity for ATP ( 90, 91), which is consistent with a shift toward the E1 conformation. Moreover, it has been reported that FXYD2 increases the K + antagonism of intracellular Na + binding, suggesting an additional effect of FXYD2 on intrinsic binding of K + at cytoplasmic sites ( 73). Finally, FXYD2 decreases the Na + activation of Na/K pump currents ( 6) and produces a parallel decrease in the Na + and K + activation of Na-K-ATPase activity ( 3).
Intriguingly, the two splice variants, FXYD2a and FXYD2b, produce identical effects on the catalytic and transport properties of Na-K-ATPase studied so far ( 6, 73). However, the functional effects of FXYD2a and FXYD2b differ depending on nonidentified posttranslational modifications, which may be cell specific or depend on the physiological state ( 2). Posttranslational modifications abolish the effect of FXYD2a on the apparent Na + affinity of Na-K-ATPase activity. On the other hand, modifications of FXYD2b do not influence the effect on the apparent Na + affinity, whereas modifications are needed for the effect of FXYD2b on the apparent K + affinity ( 2). Finally, FXYD2a, but not FXYD2b, is enriched in caveolae of renal membranes ( 31), providing another argument for different functional roles of FXYD2 variants.
The physiological relevance of Na-K-ATPase modulation by FXYD2 in the kidney remains speculative. FXYD2 are mainly distributed in renal segments, which reabsorb most of the filtered Na + load. Because a major effect of FXYD2 appears to be a decrease in the Na + affinity of Na-K-ATPase, it may be speculated that the existence of low-Na +-affinity Na-K- ATPase may be favorable for an efficient reabsorption of Na + in renal segments with high cellular Na + load ( 6). The physiological importance of the modulation of the Na + affinity of Na-K-ATPase by FXYD2 is also supported by the observation that FXYD2 transfectants in culture, which reduce Na + affinity, decrease cellular growth ( 2). On the other hand, in view of the increased affinity for ATP of Na-K-ATPase associated with FXYD2, it has been suggested that FXYD2 may be important in preserving the Na-K-ATPase activity in renal segments such as the outer medulla, which are highly prone to anoxia ( 73, 91). Significantly, renal Na-K-ATPase of FXYD2-deficient mice exhibits an increased apparent Na + affinity ( 46), confirming the relevance of the Na + effect of FXYD2 observed in different expression systems. On the other hand, mice lacking FXYD2 show no obvious defects of renal function, suggesting compensatory mechanisms that permit a normal Na + excretion balance ( 46).
Mutations in a conserved glycine residue in the TM of FXYD2 have been linked to cases of human primary hypomagnesemia ( 58). Although studies on the effect of the G/R mutation have revealed that it abolishes association of FXYD proteins with the Na-K-ATPase without a change in the cell surface expression of the Na-K-ATPase ( 23, 74), further investigations are needed to elucidate whether or how this effect may be associated with the loss of Mg 2+, increased Ca 2+ absorption, and hypocalciuria observed in these patients.
Regulation of FXYD2 expression has been studied during development and in the adult kidney. During amphibian development, FXYD2 is found in several tissues but mainly in the developing pronephric kidney ( 29). Expression of FXYD2 in the kidney coincides with the onset of expression of Na-K-ATPase 1- and 1-subunits, suggesting common regulatory mechanisms. This pattern of expression highlights the functional importance of FXYD2 in the modulation of Na-K-ATPase. In cultured murine inner medullary collecting duct (IMCD3) cells lacking FXYD2, the expression of both FXYD2a and FXYD2b is induced by hypertonicity depending on c-Jun kinase and phosphatidylinositol 3-kinase activation ( 12). Upregulated FXYD2 is routed to the basolateral membrane of IMCD3 cells, consistent with its localization in inner medullary collecting duct cells in vivo ( 72). The osmotic regulation of FXYD2 is also observed in mouse inner renal medulla in response to changes in hydration ( 12). In IMCD3 cells, failure to synthesize FXYD2 in response to hypertonicity leads to decreased cell viability, suggesting a potential role of the regulation of Na-K-ATPase by FXYD2 in cell survival under anisotonic conditions ( 12). Upregulation of FXYD2 and c-Jun kinase activation during hypertonicity is mediated by chloride but not by sodium, in contrast to upregulation of the Na-K-ATPase -subunit induced by sodium ( 14). In IMCD3 cells, activation of c-Jun kinase regulates FXYD2 at the transcriptional level, whereas activation of PI3-kinase influences the translation of FXYD2 ( 13). Finally, FXYD2a but not FXYD2b expression is induced in renal cells of proximal origin as well as in several cell lines of other than renal origin by hypertonicity, heat shock, and chemical stress ( 98). Moreover, injury-induced NF-B activation induces FXYD2 expression in the hippocampus ( 48). FXYD2a induction in cells of renal origin is accompanied by a reduction in Na-K-ATPase activity and in the rate of cell division ( 98). Both of these effects are reduced when FXYD2 expression was knocked down by small interfering RNA (siRNA), suggesting that induction of FXYD2a may be part of a cellular response to genotoxic stress ( 98).
Although much has been learned about the functional effects of FXYD2 on Na-K-ATPase and on its regulation, we are far from understanding the physiological relevance of this protein and its potential role in pathophysiological states such as human primary hypomagnesemia.
FXYD3 (Mat-8)
FXYD3 has initially been identified in murine breast tumors induced by Neu or Ras oncogenes ( 66) and was then also found to be present in primary human breast tumors and in human breast tumor cell lines ( 67), in colorectal cancer cell lines ( 101), and in prostate tumors ( 41). In normal tissue, FXYD3 is mainly expressed in the uterus, stomach, colon, and skin ( 67) ( 77). In adult mice, FXYD3 and Lgi4 genes have been mapped to the same locus on chromosome 7 and produce partially overlapping transcripts of opposite orientation, which potentially may impact on their respective expression ( 77).
When expressed in X. laevis oocytes, FXYD3 induces a hyperpolarization-activated chloride conductance similar to that observed with FXYD1 ( 67). Moreover, FXYD3 can associate with Na-K-ATPase and produces a decrease in both the apparent K + and Na + affinity of Na-K-ATPase ( 22). Interestingly, in addition to these functions shared with other FXYD proteins, FXYD3 exhibits some uncommon characteristics. First, in contrast to other FXYD proteins, FXYD3 may have two TMs because its signal sequence is not cleaved. Second, when expressed in X. laevis oocytes, FXYD3 can associate not only with Na-K-ATPase but also with gastric and colonic H-K-ATPase. However, in situ (stomach), FXYD3 is only expressed in mucous cells, which lack H-K-ATPase. Finally, after expression in X. laevis oocytes, FXYD3 modulates the processing of glycoproteins ( 22).
At present, it is difficult to draw conclusions about the physiological relevance of the Na + and K + effects of FXYD3 on Na-K-ATPase. FXYD3 is highly upregulated in specific types of cancers, such as breast or prostate tumors. Interestingly, FXYD3 has been shown to be upregulated by 5-Fluorouracil treatment of breast cancer cells in a p53-dependent way ( 57). Because p53 is an antiproliferative protein, one may assume that FXYD3 could be part of this function. However, Grzmil et al. ( 41) showed that siRNA-mediated inhibition of FXYD3 expression causes a significant reduction of cell proliferation of prostate cancer cell lines, indicating instead that FXYD3 could be involved in cell proliferation. Possibly, p53 may induce genes with proliferative functions as part of a negative feedback mechanism. It remains to be shown whether regulation of the functional properties of Na-K-ATPase, leading to modifications of the cellular milieu, is important in impeding or triggering cell proliferation. Finally, because FXYD3 not only colocalizes with Na-K-ATPase in basolateral membranes but is also found in apical membranes of mucous cells of the stomach ( 22), it cannot be excluded that FXYD3 is not only a Na-K-ATPase modulator but also has other yet unknown functions.
FXYD4 (CHIF)
FXYD4 is expressed in kidney medullary collecting duct and papilla and in the distal colon ( 16, 81) ( Table 2). Like FXYD2, FXYD4 is only expressed in basolateral membranes of target epithelia.
Because FXYD4 induces a depolarization-dependent K +-specific ion conductance when expressed in X. laevis oocytes, it has been suggested that FXYD4 may be a regulator of ion channels ( 5). However, this channel activity turned out to be irreproducible ( 81), and it is more likely that, as suggested by more recent evidence, FXYD4 associates specifically with Na-K-ATPase in expression systems and in situ, as well as modulates its transport properties ( 6, 37).
In the absence of extracellular Na +, a situation in which K + kinetics most closely reflect the intrinsic affinity for extracellular K +, FXYD4 has no effect on the K + activation of Na-K-ATPase. On the other hand, in the presence of extracellular Na +, a condition in which the K + kinetics depend on the competition by extracellular Na +, FXYD4 produces a large increase in the K1/2 value for K + of the Na-K-ATPase ( 6). Moreover, FXYD4 induces a two- to threefold increase in the apparent affinity for intracellular Na + ( 6, 37). This Na + effect of FXYD4 on Na-K-ATPase is likely to be of physiological relevance for the Na + reabsorption process in the collecting duct, which is the ultimate site of electrolyte conservation. An increase in the Na + affinity of Na-K-ATPase produced by the association with FXYD4 in this renal segment is favorable because it permits efficient Na + reabsorption even at low intracellular Na + concentrations. At physiologically low intracellular Na + concentrations (e.g., 5 mM), the Na-K-ATPase transport rate of Na-K-ATPase associated with FXYD4 should be about four times higher than that of Na-K-ATPase lacking FXYD4 ( 6).
Results from FXYD4 knockout mice ( 1, 40) at least partially confirm that a major if not unique effect of FXYD4 is the activation of Na-K-ATPase by increasing its apparent Na + affinity. Abolition of FXYD4 expression appears to be fully compensated for in the kidney because fractional excretions of Na + and K + are normal under resting conditions, and animals have no deficit in the adaptation to low Na + and K + intake. However, in the distal colon, amiloride-inhibitable Na + reabsorption is reduced under control conditions, glucocorticoid treatment, and low Na + intake ( 40).
Regulation of FXYD4 expression in the kidney and colon is different. Both distal colon and the renal collecting duct, where FXYD4 is expressed, are aldosterone target sites, but aldosterone induces FXYD4 cRNA expression only in the colon but not in the kidney ( 11, 97). On the other hand, low Na + intake increases FXYD4 mRNA and protein expression in the colon, but only FXYD4 protein and not mRNA expression in the kidney ( 16, 81). Moreover, in experimental ischemic acute renal failure ( 39, 75) and in acute tubular necrosis ( 84), which are characterized by hyperkalemia, FXYD4 cRNA expression is decreased in kidney and increased in colon. Possibly, suppression of FXYD4 expression in kidney could lead to decreased renal K + secretion, and the enhanced FXYD4 expression in colon might be an adaptive response.
Although compelling evidence suggests that an important biological activity of FXYD4 is mediated through its interaction with Na-K-ATPase, it remains open whether this is the only function of FXYD4 or whether FXYD4 interacts with and regulates other, as yet unidentified, partner proteins.
FXYD5 (Ric) AND FXYD6 (PHOSPHOHIPPOLIN)
FXYD5 ( 34) and FXYD6 ( 100) are poorly characterized, and interaction with and modulation of Na-K-ATPase have so far not been reported.
Compared with other FXYD proteins, FXYD5 has an unusual long NH 2-terminal extension ( 86). The so-called IUW-1 protein, which may be an isoform of FXYD5, has a shorter NH 2 terminus and shows 61% sequence identity with the cytoplasmic domain of the angiotensin II type 1 receptor ( 70). FXYD5 expression is induced in cells transformed by the oncogene E2a-Pbxl ( 34). FXYD5 is also expressed in several cancer tissues but only in a few normal cell types ( 44). A role of FXYD5 in tumor progression and metastasis has been suggested, based on the observations that transfection of FXYD5, called dysadherin, into liver cancer cells results in decreased E-cadherin-mediated cell-cell adhesion ( 44), implicating O-glycosylation of FXYD5 ( 94). Moreover, dysadherin positivity was correlated with poor prognosis in various human cancers ( 82, 83).
FXYD6 is expressed in several tissues ( 86). In the brain, expression studies during development suggest a role in neuronal excitability during postnatal development and in the adult brain ( 47, 78).
FXYD7
FXYD7 has been found exclusively in the brain, where it is expressed in neurons and to a lesser extent in glial cells ( 7). FXYD7 is subjected to O-glycosylation, which appears to be necessary for the stability of the protein ( 7).
Mutational analysis of FXYD7-specific regions revealed a COOH-terminal, cytoplasmic valine residue that is involved in rapid endoplasmic reticulum export and controls the rate of its cell surface expression ( 23). After coexpression in X. laevis oocytes, FXYD7 associates posttranslationally with Na-K-ATPase ( 23), probably due to the different rates of endoplasmic reticulular exit of the two proteins. Association of FXYD7 occurs with 1 1, 2 1, and 3 1 isozymes but not with Na-K-ATPase isozymes containing 2-isoforms ( 7). On the other hand, in the brain, FXYD7 exclusively associates with Na-K-ATPase containing 1-isoforms ( 7). These data suggest that in the brain, FXYD7 is specifically associated with Na-K-ATPase 1 1-isozymes.
Electrophysiological analysis of the modulatory effect of FXYD7 on Na-K-ATPase in X. laevis oocytes revealed that FXYD7 modulates the transport properties of 1 1-isozymes in a specific way, distinct from other FXYD proteins ( 7). The apparent affinity for K + is increased over a wide range of membrane potentials in both the presence and absence of extracellular Na +, which suggests a modification of the intrinsic affinity of the external K +-binding site. On the other hand, FXYD7 does not influence the apparent affinity for intracellular Na + ( 7). It can be speculated that in the brain, the existence of Na-K-ATPase 1 1-isozymes with low K + affinity, acquired by association with FXYD7, may be necessary for efficient clearance of extracellular K + during neuronal activity to ensure neuronal excitability.
Future studies should be directed to determine whether the endoplasmic reticular export control of FXYD7 dependent on the COOH-terminal valine residue may be linked to the specific expression of FXYD7 in neurons and glial cells and/or to the particular requirements of the regulation of Na-K-ATPase expression and function in the brain.
STRUCTURE-FUNCTION RELATIONSHIP
In addition to the elucidation of the functional effects on Na-K-ATPase of several FXYD proteins, recent studies have revealed details on the molecular basis of these effects and on the structural and functional interaction sites in Na-K-ATPase and FXYD proteins. It is becoming clear that multiple sites of interaction exist that involve both the transmembrane and extramembrane domains.
The role of conserved amino acids in the structural and functional interaction with Na-K-ATPase has been studied in some FXYD proteins. Replacement of Gly-41 with Arg in the TM domain of FXYD2 ( 103), which is associated with a form of renal hypomagnesemia ( 58), or replacement of the analogous Gly-40 with Arg in FXYD7 ( 23) abolishes the interaction of these FXYD proteins with Na-K-ATPase. Replacements with Leu of Gly-41 in FXYD2 ( 103) or with Ala or Trp of Gly-40 in FXYD7 ( 23) permit partial association with Na-K-ATPase, which is paralleled by a partial loss of the effect of FXYD7 on the apparent K + affinity of Na-K-ATPase ( 23). A similar reduction of the association efficiency and the K + effect is also observed when the other conserved Gly-29 in FXYD7 is replaced by Ala. Gly-40 and Gly-29 are on the same side of the TM -helix of FXYD proteins and form a groove that could be important as an interaction site between Na-K-ATPase and FXYD proteins. On the other hand, experiments using synthetic transmembrane mimetic peptides suggest that Gly-41 in FXYD2 is important for the effect of FXYD2 on the apparent Na + affinity of Na-K-ATPase and not primarily on the association of FXYD2 with Na-K-ATPase ( 103). Finally, experiments using perfluorooctanoate gel electrophoresis indicate that FXYD2 and FXYD1 ( 56) and peptides corresponding to the TM domain of FXYD2 ( 89) may form oligomers. Gly-41 is necessary for oligomerization of FXYD2 peptides ( 89).
The role of the conserved FXYD motif has been studied in several FXYD proteins, but so far no preserved function could be revealed. The FXYD motif is important for the stable interaction of Na-K-ATPase with FXYD2 and FXYD4 ( 6) but not with FXYD7 ( 23).
Because an antibody against the COOH terminus of FXYD2 ( 73, 91) and truncations of the COOH and the NH 2 termini of FXYD2 ( 74) abolish the effect of FXYD2 on the apparent affinity for ATP of Na-K-ATPase, it was concluded that extramembrane interactions may be important for this functional effect of FXYD2. Interestingly, in FXYD2-deficient mice, the ATP affinity of Na-K-ATPase was not decreased as expected, but slightly increased, suggesting that extramembrane interactions may be context specific ( 46). On the other hand, by examining FXYD2/FXYD4 chimera, it was found that TM interactions of FXYD proteins determine both the stability of FXYD proteins in detergents and the effects of these FXYD proteins on the Na + affinity of Na-K-ATPase ( 52). Interestingly, different amino acids in the TM appear to mediate the stability and the functional effects of these FXYD proteins. The functional role of TM interactions in the Na + effect of FXYD2 was confirmed by showing that peptides corresponding to the TM domain of FXYD2 decrease the Na + affinity of Na-K-ATPase similarly to full-length FXYD2 ( 103).
Recent experimental evidence also shed some light on the sites where FXYD proteins interact with Na-K-ATPase / complexes. Based on experiments using thermal denaturation, it has been suggested that FXYD2 interacts with TM8–10 of the Na-K-ATPase -subunit ( 27). Moreover, electron crystallographic analysis of renal Na-K-ATPase at 9.5- resolution ( 42) and taking as a basis the high-resolution structure of the Ca-ATPase ( 93) suggest that FXYD2 is located in a binding pocket made up of TM9, TM6, TM4, and TM2. A role of TM9 in the structural and functional interaction with FXYD proteins could be confirmed by mutational analysis ( 51). Interestingly, Leu 964 and Phe 967 of TM9 of the rat Na-K-ATPase contribute to the stable interaction with FXYD2, FXYD4, and FXYD7 but do not influence the functional effect of these FXYD proteins on the apparent K + affinity of Na-K-ATPase. On the other hand, Phe 956 and Glu 960 do not contribute to the efficient association of FXYD proteins but transmit the K + effect of FXYD proteins to Na-K-ATPase. The relative contribution of Phe 956 and Glu 960 in the K + effect of different FXYD proteins is different, probably reflecting the intrinsic variations in the modulation of the apparent K + affinity of Na-K-ATPase by different FXYD proteins. In contrast to the K + effect, the modulation of the apparent Na + affinity of Na-K-ATPase by FXYD4 is not mediated by Phe 956 and Glu 960. The mutational analysis is in good agreement with a docking model of the Na-K-ATPase -subunit/FXYD7 complex ( 51). Thus the structural and functional interaction with FXYD proteins involve TM9 of the Na-K-ATPase -subunit but probably also other TM domains that remain to be determined. Based on cross-linking data, interactions of FXYD2 with the intracellular loop L6/7 in the Na-K-ATPase -subunit and with the extracellular domain of the Na-K-ATPase -subunit can also be predicted ( 36). Moreover, modeling of Na-K-ATPase/FXYD2 complexes is in agreement with the position of FXYD2 in a binding pocket composed of TM9, TM6, TM4, and TM2.
In conclusion, recent experimental evidence strongly supports that at least one functional role of FXYD proteins is the regulation of Na-K-ATPase in a tissue- and isoform-specific way. Although the functional effects of FXYD proteins on the transport and kinetic properties of Na-K-ATPase have been well characterized, much still remains to be learned about the physiological relevance of these effects and the potential implication of a loss of FXYD regulation of Na-K-ATPase in pathophysiological states. For a better understanding of these issues, more studies are needed on the functional roles of each FXYD protein, on their biosynthesis and processing, their tissue and cellular distribution, and their structure-function relationship.
GRANTS
This work was supported by Swiss National Fund Grant 3100A0–107513/1 (to K. Geering).
FOOTNOTES
REFERENCES
Aizman R, Asher C, Fuzesi M, Latter H, Lonai P, Karlish SJD, and Garty H. Generation and phenotypic analysis of CHIF knockout mice. Am J Physiol Renal Physiol 283: F569–F577, 2002.
Arystarkhova E, Donnet C, Asinovski NK, and Sweadner KJ. Differential regulation of renal Na,K-ATPase by splice variants of the subunit. J Biol Chem 277: 10162–10172, 2002.
Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. The subunit modulates Na + and K + affinity of the renal Na,K-ATPase. J Biol Chem 274: 33183–33185, 1999.
Arystarkhova E, Wetzel RK, and Sweadner KJ. Distribution and oligomeric association of splice forms of Na +-K +-ATPase regulatory -subunit in rat kidney. Am J Physiol Renal Physiol 282: F393–F407, 2002.
Attali B, Latter H, Rachamim N, and Garty H. A corticosteroid-induced gene expressing an "IsK-like" K + channel activity in Xenopus oocytes. Proc Natl Acad Sci USA 92: 6092–6096, 1995.
Beguin P, Crambert G, Guennoun S, Garty H, Horisberger JD, and Geering K. CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the -subunit. EMBO J 20: 3993–4002, 2001.
Beguin P, Crambert G, Monnet-Tschudi F, Uldry M, Horisberger JD, Garty H, and Geering K. FXYD7 is a brain-specific regulator of Na,K-ATPase 1- isozymes. EMBO J 21: 3264–3273, 2002.
Beguin P, Wang XY, Firsov D, Puoti A, Claeys D, Horisberger JD, and Geering K. The subunit is a specific component of the Na,K-ATPase and modulates its transport properties. EMBO J 16: 4250–4260, 1997.
Blanco G and Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 275: F633–F650, 1998.
Bogaev RC, Jia LG, Kobayashi YM, Palmer CJ, Mounsey JP, Moorman JR, Jones LR, and Tucker AL. Gene structure and expression of phospholemman in mouse. Gene 271: 69–79, 2001.
Brennan FE and Fuller PJ. Acute regulation by corticosteroids of channel-inducing factor gene messenger ribonucleic acid in the distal colon. Endocrinology 140: 1213–1218, 1999.
Capasso JM, Rivard C, and Berl T. The expression of the subunit of Na-K-ATPase is regulated by osmolality via C-terminal Jun kinase and phosphatidylinositol 3-kinase-dependent mechanisms. Proc Natl Acad Sci USA 98: 13414–13419, 2001.
Capasso JM, Rivard CJ, and Berl T. Synthesis of the Na-K-ATPase -subunit is regulated at both the transcriptional and translational levels in IMCD3 cells. Am J Physiol Renal Physiol 288: F76–F81, 2005.
Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Chloride, not sodium, stimulates expression of the subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells. Proc Natl Acad Sci USA 100: 6428–6433, 2003.
Capendeguy O and Horisberger JD. Functional effects of Na +,K +-ATPase gene mutations. Neuromol Med 6: 105–116, 2004.
Capurro C, Coutry N, Bonvalet JP, Escoubet B, Garty H, and Farman N. Cellular localization and regulation of CHIF in kidney and colon. Am J Physiol Cell Physiol 271: C753–C762, 1996.
Chen Z, Jones LR, O'Brian JJ, Moorman JR, and Cala SE. Structural domains in phospholemman: a possible role for the carboxyl terminus in channel inactivation. Circ Res 82: 367–374, 1998.
Cornelius F and Mahmmoud YA. Functional modulation of the sodium pump: the regulatory proteins "Fixit." News Physiol Sci 18: 119–124, 2003.
Crambert G, Fuzesi M, Garty H, Karlish S, and Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci USA 99: 11476–11481, 2002.
Crambert G and Geering K. FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. Sci STKE 166: RE1, 2003.
Crambert G, Hasler U, Beggah AT, Yu C, Modyanov NN, Horisberger JD, Lelievre L, and Geering K. Transport and pharmacological properties of nine different human Na,K-ATPase isozymes. J Biol Chem 275: 1976–1986, 2000.
Crambert G, Li C, Claeys D, and Geering K. FXYD3 (Mat-8), a new regulator of Na,K-ATPase. Mol Biol Cell 16: 2363–2371, 2005.
Crambert G, Li C, Swee LK, and Geering K. FXYD7: mapping of functional sites involved in endoplasmic reticulum export, association with and regulation of Na,K-ATPase. J Biol Chem 279: 30888–30895, 2004.
Crowell KJ, Franzin CM, Koltay A, Lee S, Lucchese AM, Snyder BC, and Marassi FM. Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers. Biochim Biophys Acta 1645: 15–21, 2003.
De Carvalho Aguiar P, Sweadner KJ, Penniston JT, Zaremba J, Liu L, Caton M, Linazasoro G, Borg M, Tijssen MA, Bressman SB, Dobyns WB, Brashear A, and Ozelius LJ. Mutations in the Na +/K +-ATPase 3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism. Neuron 43: 169–175, 2004.
De Fusco M, Marconi R, Silvestri L, Atorino L, Rampoldi L, Morgante L, Ballabio A, Aridon P, and Casari G. Haploinsufficiency of ATP1A2 encoding the Na +/K + pump 2 subunit associated with familial hemiplegic migraine type 2. Nat Genet 33: 192–196, 2003.
Donnet C, Arystarkhova E, and Sweadner KJ. Thermal denaturation of the Na,K-ATPase provides evidence for - oligomeric interaction and subunit association with the C-terminal domain. J Biol Chem 276: 7357–7365, 2001.
Dostanic I, Schultz JEJ, Lorenz JN, and Lingrel JB. The 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart. J Biol Chem 279: 54053–54061, 2004.
Eid SR and Brandli AW. Xenopus Na,K-ATPase: primary sequence of the 2 subunit and in situ localization of 1, 1, and expression during pronephric kidney development. Differentiation 68: 115–125, 2001.
Feraille E and Doucet A. Sodium-potassium-adenosine triphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345–418, 2001.
Ferrandi M, Molinari I, Barassi P, Minotti E, Bianchi G, and Ferrari P. Organ hypertrophic signaling within caveolae membrane subdomains triggered by ouabain and antagonized by PST 2238. J Biol Chem 279: 33306–33314, 2004.
Feschenko MS, Donnet C, Wetzel RK, Asinovski NK, Jones LR, and Sweadner KJ. Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus. J Neurosci 23: 2161–2169, 2003.
Forbush B III, Kaplan JH, and Hoffman JF. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry 17: 3667–3676, 1978.
Fu X and Kamps M. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts. Mol Cell Biol 17: 1503–1512, 1997.
Fuller W, Eaton P, Bell JR, and Shattock MJ. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K ATPase. FASEB J 18: 197–199, 2003.
Fuzesi M, Gottschalk KE, Lindzen M, Shainskaya A, Kuster B, Garty H, and Karlish SJ. Covalent cross-links between the subunit(FXYD2) and and subunits of Na,K-ATPase. Modeling the - interaction. J Biol Chem 280: 18291–18301, 2005.
Garty H, Lindzen M, Scanzano R, Aizman R, Fuzesi M, Goldshleger R, Farman N, Blostein R, and Karlish SJD. A functional interaction between CHIF and Na-K-ATPase: implication for regulation by FXYD proteins. Am J Physiol Renal Physiol 283: F607–F615, 2002.
Geering K. The functional role of subunits in oligomeric P-type ATPases. J Bioenerg Biomembr 33: 425–438, 2001.
Gimelreich D, Popovtzer MM, Wald H, Pizov G, Berlatzky Y, and Rubinger D. Regulation of ROMK and channel-inducing factor (CHIF) in acute renal failure due to ischemic reperfusion injury. Kidney Int 59: 1812–1820, 2001.
Goldschimdt I, Grahammer F, Warth R, Schulz-Baldes A, Garty H, Greger R, and Bleich M. Kidney and colon electrolyte transport in CHIF knockout mice. Cell Physiol Biochem 14: 113–120, 2004.
Grzmil M, Voigt S, Thelen P, Hemmerlein B, Helmke K, and Burfeind P. Up-regulated expression of the MAT-8 gene in prostate cancer and its siRNA-mediated inhibition of expression induces a decrease in proliferation of human prostate carcinoma cells. Int J Oncol 24: 97–105, 2004.
Hebert H, Purhonen P, Vorum H, Thomsen K, and Maunsbach AB. Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals. J Mol Biol 314: 479–494, 2001.
Horisberger JD. Recent insights into the structure and mechanism of the sodium pump. Physiology (Bethesda) 19: 377–387, 2004.
Ino Y, Gotoh M, Sakamoto M, Tsukagoshi K, and Hirohashi S. Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis. Proc Natl Acad Sci USA 99: 365–370, 2002.
Jia LG, Donnet C, Bogaev RC, Blatt RJ, McKinney CE, Day KH, Berr SS, Jones LR, Moorman JR, Sweadner KJ, and Tucker AL. Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice. Am J Physiol Heart Circ Physiol 288: H1982–H1988, 2005.
Jones DH, Li TY, Arystarkhova E, Barr KJ, Wetzel RK, Peng J, Markham K, Sweadner KJ, Fong GH, and Kidder GM. Na,K-ATPase from mice lacking the subunit (FXYD2) exhibits altered Na + affinity and decreased thermal stability. J Biol Chem 280: 19003–19011, 2005.
Kadowaki K, Sugimoto K, Yamaguchi F, Song T, Watanabe Y, Singh K, and Tokuda M. Phosphohippolin expression in the rat central nervous system. Mol Brain Res 125: 105–112, 2004.
Kassed CA, Butler TL, Patton GW, Demesquita DD, Navidomskis MT, Memet S, Israel A, and Pennypacker KR. Injury-induced NF-B activation in the hippocampus: implications for neuronal survival. FASEB J 18: 723–724, 2004.
Kuster B, Shainskaya A, Pu HX, Goldshleger R, Blostein R, Mann M, and Karlish SJ. A new variant of the subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells. J Biol Chem 275: 18441–18446, 2000.
Laski ME and Kurtzman NA. The renal adenosine triphosphatases—functional integration and clinical significance. Miner Electrolyte Metab 22: 410–422, 1996.
Li C, Grosdidier A, Crambert G, Horisberger JD, Michielin O, and Geering K. Structural and functional interaction sites between Na,K-ATPase and FXYD proteins. J Biol Chem 279: 38895–38902, 2004.
Lindzen M, Aizman R, Lifshitz Y, Lubarski I, Karlish SJD, and Garty H. Structure-function relations of interactions between Na,K-ATPase, the subunit, and corticosteroid hormone-induced factor. J Biol Chem 278: 18738–18743, 2003.
Lu KP, Kemp BE, and Means AR. Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J Biol Chem 269: 6603–6607, 1994.
Mahmmoud YA and Cornelius F. Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the - and -subunits close to the inner face of the plasma membrane. Biophys J 82: 1907–1919, 2002.
Mahmmoud YA, Cramb G, Maunsbach AB, Cutler CP, Meischke L, and Cornelius F. Regulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS. J Biol Chem 278: 37427–37438, 2003.
Mahmmoud YA, Vorum H, and Cornelius F. Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase C via a novel member of the FXYDY family. J Biol Chem 275: 35969–35977, 2000.
Maxwell PJ, Longley DB, Latif T, Boyer J, Allen W, Lynch M, McDermott U, Harkin DP, Allegra CJ, and Johnston PG. Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling. Cancer Res 63: 4602–4606, 2003.
Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, de Pont JJ, Bindels RJ, Monnens LA, van den Heuvel LP, and Knoers NV. Dominant isolated renal magnesium loss is caused by misrouting of the Na +,K +-ATPase -subunit. Nat Genet 26: 265–266, 2000.
Mercer RW, Biemesderfer D, Bliss DP, Collins JH, and Forbush B. Molecular cloning and immunological characterization of the -polypeptide, a small protein associated with the Na,K-ATPase. J Cell Biol 121: 579–586, 1993.
Mirza MA, Zhang XQ, Ahlers BA, Qureshi A, Carl LL, Song J, Tucker AL, Mounsey JP, Moorman JR, Rothblum LI, Zhang TS, and Cheung JY. Effects of phospholemman downregulation on contractility and Ca 2+i transients in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H1322–H1330, 2004.
Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, and Bennett V. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421: 634–639, 2003.
Moorman JR, Ackerman SJ, Kowdley GC, Griffin MP, Mounsey JP, Chen ZH, Cala SE, Obrian JJ, Szabo G, and Jones LR. Unitary anion currents trough phospholemman channel molecules. Nature 377: 737–740, 1995.
Moorman JR, Palmer CJ, John JE III, Durieux ME, and Jones LR. Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes. J Biol Chem 267: 14551–14554, 1992.
Morales-Mulia M, Pasantes-Morales H, and Moran J. Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman. Biochim Biophys Acta 1496: 252–260, 2000.
Moran J, Morales-Mulia M, and Pasantes-Morales H. Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes. Biochim Biophys Acta 1538: 313–320, 2001.
Morrison BW and Leder P. Neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors. Oncogene 9: 3417–3426, 1994.
Morrison BW, Moorman JR, Kowdley GC, Kobayashi YM, Jones LR, and Leder P. Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. J Biol Chem 270: 2176–2182, 1995.
Mounsey JP, John JE III, Helmke SM, Bush EW, Gilbert J, Roses AD, Perryman MB, Jones LR, and Moorman JR. Phospholemman is a substrate for myotonic dystrophy protein kinase. J Biol Chem 275: 23362–2367, 2000.
Mounsey JP, Lu KP, Patel MK, Chen ZH, Horne LT, John JE, Means AR III, Jones LR, and Moorman JR. Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases. Biochim Biophys Acta 1451: 305–318, 1999.
Omasa T, Chen YG, Mantalaris A, and Wu JH. A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP11/PLM/MAT8 family of transmembrane proteins. Biochim Biophys Acta 1517: 307–310, 2001.
Palmer CJ, Scott BT, and Jones LR. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 266: 11126–11130, 1991.
Pihakaski-Maunsbach K, Tokonabe S, Vorum H, Rivard CJ, Capasso JM, Berl T, and Maunsbach AB. The -subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity. Am J Physiol Renal Physiol 288: F650–F657, 2005.
Pu HX, Cluzeaud F, Goldshlegger R, Karlish SJD, Farman N, and Blostein R. Functional role and immunocytochemical localization of the a and b forms of the Na,K-ATPase subunit. J Biol Chem 276: 20370–20378, 2001.
Pu HX, Scanzano R, and Blostein R. Distinct regulatory effects of the Na,K-ATPase subunit. J Biol Chem 277: 20270–20276, 2002.
Rabb H, Wang Z, Postler G, and Soleimani M. Possible molecular basis for changes in potassium handling in acute renal failure. Am J Kidney Dis 35: 871–877, 2000.
Rose AM and Valdes R. Understanding the sodium pump and its relevance to disease. Clin Chem 40: 1674–1685, 1994.
Runkel F, Michels M, and Franz T. Fxyd3 and Lgi4 expression in the adult mouse: a case of endogenous antisense expression. Mamm Genome 14: 665–672, 2003.
Saito S, Matoba R, Kato K, and Matsubara K. Expression of a novel member of the ATP11/PLM/MAT8 family, phospholemman-like protein (PLP) gene, in the developmental process of mouse cerebellum. Gene 279: 149–155, 2001.
Segall L, Scanzano R, Kaunisto MA, Wessman M, Palotie A, Gargus JJ, and Blostein R. Kinetic alterations due to a missense mutation in the Na,K-ATPase 2 subunit cause familial hemiplegic migraine type 2. J BiolChem 279: 43692–43696, 2004.
Sha Q, Lansbery KL, Distefano D, Mercer RW, and Nichols CG. Heterologous expression of the Na +,K +-ATPase subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore. J Physiol 535: 407–417, 2001.
Shi H, Levy-Holzman R, Cluzeaud F, Farman N, and Garty H. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505–F512, 2001.
Shimada Y, Yamasaki S, Hashimoto Y, Ito T, Kawamura J, Soma T, Ino Y, Nakanishi Y, Sakamoto M, Hirohashi S, and Imamura M. Clinical significance of dysadherin expression in gastric cancer patients. Clin Cancer Res 10: 2818–2823, 2004.
Shimamura T, Sakamoto M, Ino Y, Sato Y, Shimada K, Kosuge T, Sekihara H, and Hirohashi S. Dysadherin overexpression in pancreatic ductal adenocarcinoma reflects tumor aggressiveness: relationship to E-cadherin expression. J Clin Oncol 21: 659–667, 2003.
Shustin L, Wald H, and Popovtzer MM. Role of down-regulated CHIF mRNA in the pathophysiology of hyperkalemia of acute tubular necrosis. Am J Kidney Dis 32: 600–604, 1998.
Silverman BdZ, Fuller W, Eaton P, Deng J, Moorman JR, Cheung JY, James AF, and Shattock MJ. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovasc Res 65: 93–103, 2005.
Sweadner KJ and Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68: 41–56, 2000.
Sweadner KJ, Wetzel RK, and Arystarkhova E. Genomic organization of the human FXYD2 gene encoding the of the Na,K-ATPase. Biochem Biophys Res Commun 279: 196–201, 2000.
Therien AG and Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541–C566, 2000.
Therien AG and Deber CM. Oligomerization of a peptide derived from the transmembrane region of the sodium pump subunit: effect of the pathological mutation G41R. J Mol Biol 322: 583–590, 2002.
Therien AG, Goldshleger R, Karlish SJ, and Blostein R. Tissue-specific distribution and modulatory role of the subunit of the Na,K-ATPase. J Biol Chem 272: 32628–32634, 1997.
Therien AG, Karlish SJ, and Blostein R. Expression and functional role of the subunit of the Na,K-ATPase in mammalian cells. J Biol Chem 274: 12252–12256, 1999.
Therien AG, Pu HX, Karlish SJ, and Blostein R. Molecular and functional studies of the subunit of the sodium pump. J Bioenerg Biomembr 33: 407–414, 2001.
Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405: 647–655, 2000.
Tsuiji H, Takasaki S, Sakamoto M, Irimura T, and Hirohashi S. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell-cell adhesion. Glycobiology 13: 521–527, 2003.
Vanmolkot KR, Kors EE, Hottenga JJ, Terwindt GM, Haan J, Hoefnagels WA, Black DF, Sandkuijl LA, Frants RR, Ferrari MD, and van den Maagdenberg AM. Novel mutations in the Na +,K +-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions. Ann Neurol 54: 360–366, 2003.
Walaas SI, Czernik AJ, Olstad OK, Sletten K, and Walaas O. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem J 304: 635–640, 1994.
Wald H, Goldstein O, Asher C, Yagil Y, and Garty H. Aldosterone induction and epithelial distribution of CHIF. Am J Physiol Renal Fluid Electrolyte Physiol 271: F322–F329, 1996.
Wetzel RK, Pascoa JL, and Arystarkhova E. Stress-induced expression of the subunit (FXYD2) modulates Na,K-ATPase activity and cell growth. J Biol Chem 279: 41750–41757, 2004.
Wetzel RK and Sweadner KJ. Phospholemman expression in extraglomerular mesangium and afferent arteriole of the juxtaglomerular apparatus. Am J Physiol Renal Physiol 285: F121–F129, 2003.
Yamaguchi F, Yamaguchi K, Tai Y, Sugimoto K, and Tokuda M. Molecular cloning and characterization of a novel phospholemman-like protein from rat hippocampus. Brain Res Mol Brain Res 86: 189–192, 2001.
Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, and Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 96: 14517–14522, 1999.
Zhang XQ, Qureshi A, Song J, Carl LL, Tian Q, Stahl RC, Carey DJ, Rothblum LI, and Cheung JY. Phospholemman modulates Na +/Ca 2+ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 284: H225–H233, 2003.
Zouzoulas A, Therien AG, Scanzano R, Deber CM, and Blostein R. Modulation of Na,K-ATPase by the subunit: studies with transfected cells and transmembrane mimetic peptides. J Biol Chem 278: 40437–40441, 2003.
ABSTRACT
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 (-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.
ion transport regulation; protein-protein interaction; structure-function relationship
THE NA-K-ATPASE, OR NA/K PUMP, belongs to the protein family of P-type ATPases that are characterized by the formation of a phosphorylated intermediate during the catalytic cycle. By using the energy of the hydrolysis of ATP, the Na-K-ATPase, located in the plasma membrane, transports three Na + ions out of the cell in exchange for two K + ions into the cell ( 43). The Na-K-ATPase is an oligomeric protein. The catalytic -subunit with 10 transmembrane segments transports the cations, hydrolyzes ATP, and is the pharmacological receptor for cardiac glycosides. A type II, glycosylated -subunit functions as a molecular chaperone necessary for the correct membrane insertion of the -subunit during its synthesis and modulates its transport properties ( 38). Four - and three -isoforms have been identified, which show a tissue-specific expression and can potentially form 12 different Na-K-ATPase isozymes with distinct transport and pharmacological properties ( 9, 21).
The Na-K-ATPase is the transport system responsible for the maintenance of the Na + and K + gradients across the plasma membrane. These gradients are essential to maintain cell volume and membrane potential, and the Na + gradients provide the energy for the activity for secondary Na +-dependent transport systems that supply the cell with nutrients and regulate intracellular pH and Ca 2+ concentrations. The Na-K-ATPase is present in all cells to ensure basic cellular homeostasis but it also contributes to specialized tissue functions. In muscle cells, the activity of Na-K-ATPase is tightly coupled to the activity of a Na/Ca exchanger and thus favors contractility of heart and skeletal muscles. The presence of Na-K-ATPase in nerve and glial cells permits restoration of the Na + and K + gradients during neuronal activity and thus ensures neuronal excitability. Moreover, in the kidney Na-K-ATPase is exclusively located in the basolateral membrane of epithelial cells and thus becomes the driving force for Na + reabsorption essential to maintain extracellular volume and blood pressure.
In view of its important physiological role, it may be expected that dysfunction or dysregulation of the Na-K- ATPase may have severe pathophysiological consequences. However, only recently have mutations in Na-K-ATPase been associated with genetic diseases. Four different mutations in a single allele of the 2-isoform of the Na-K-ATPase, which cause loss of function, are linked to familial hemiplegic migraine type 2 (FHM2), a hereditary form of migraine characterized by aura and some hemiparesis ( 15, 26, 79, 95). Moreover, missense mutations in the gene for the 3-isoform of the Na-K-ATPase have been identified as a cause of rapid-onset dystonia-Parkinsonism (RDP; DYT12) ( 25). Dysregulation of Na-K-ATPase through a defective production or function of a tissue-specific regulator has been correlated with various disorders, including cardiovascular, neurological, renal, and metabolic diseases, but the direct link between altered pump function and defective regulation remains in most cases obscure ( 50, 76).
Numerous mechanisms are involved in the regulation of the Na-K-ATPase to adapt its activity and/or expression to changing physiological demands, permitting proper "housekeeping" and specialized tissue functions of Na-K-ATPase. The requirements for Na-K-ATPase modulation are likely to be greatest in response to physiological stimuli such as nerve impulse propagation, exercise, and changes in dietary Na + and K +.
Because intracellular Na + is the limiting factor for Na-K-ATPase activity, any change in the intracellular Na + concentration affects its transport rate. Moreover, peptide hormones or neurotransmitters may provoke phosphorylation of Na-K- ATPase by protein kinases, which modulates its cell surface expression ( 88). Finally, steroid hormones such as aldosterone affect and gene transcription, leading to an increased number of Na-K-ATPase pump units ( 30).
In addition to these regulatory effects mediated by hormones and neurotransmitters, recent experimental evidence has revealed a novel regulatory mechanism that involves interaction of the Na-K-ATPase with small-membrane proteins of the FXYD family. In contrast to hormonal regulation, interaction of FXYD proteins does not produce a change in Na-K-ATPase expression but rather modifies the transport properties of the Na-K-ATPase in a tissue- and isoform-specific way.
THE FXYD PROTEIN FAMILY
Sweadner and Rael ( 86) have defined the gene family of FXYD proteins based on the invariant amino acids in a signature sequence containing the FXYD motif and two conserved glycine and a serine residue. In mammals, this family contains seven members including FXYD1 (or phospholemman) ( 71); FXYD2 (or the -subunit of Na-K-ATPase) ( 33, 59); FXYD3 (or mammary tumor marker Mat-8) ( 67); FXYD4 (or corticosteroid hormone-induced factor CHIF) ( 5); FXYD5 (or related to ion channel RIC or dysadherin) ( 34); FXYD6 (or phosphohippolin) ( 100); and FXYD7 ( 7) ( Fig. 1). Moreover, FXYD2 from Xenopus laevis ( 8) and a phospholemman-like protein from shark ( 55) have been cloned. FXYD proteins contain 61–95 amino acids, with the exception of FXYD5, which has 178 amino acids due to an NH 2-terminal extension. FXYD1 ( 71), FXYD2 ( 8), FXYD4 ( 6), and FXYD7 ( 7) are type I membrane proteins with a single membrane span and the COOH terminus exposed to the cytosol. FXYD1 and FXYD4 achieve this membrane orientation after cleavage of a signal peptide, whereas FXYD2 and FXYD7 have no signal peptide. Circular dichroism and NMR spectra predict that the transmembrane domains (TMs) of FXYD1, 2, 3, and 4 adopt an -helical conformation ( 24, 89). For years after their cloning, the functional role of FXYD proteins remained unknown. Because FXYD1 ( 63), FXYD3 ( 67), FXYD4 ( 5), and FXYD5 ( 34) induce ion-specific conductances when overexpressed in X. laevis, it was long thought that FXYD proteins may be ion channels or regulators of ion channels. However, the physiological significance of these observations remains unclear because induction of ion conductances was often observed only under nonphysiological conditions. Experimental evidence then showed that FXYD2, known for over 20 years to be associated with renal Na-K-ATPase ( 33), indeed modulates Na-K-ATPase activity ( 8). This promoted intense research that showed, until now, that at least five of the seven FXYD proteins interact with Na-K-ATPase and regulate its function. Thus, even though it is not known whether Na-K-ATPase regulation is the only function of FXYD proteins, it is likely that all FXYD proteins are tissue-specific auxiliary subunits of Na-K-ATPase. The experimental criteria that have been used to support this hypothesis are 1) coimmunoprecipitation of FXYD proteins with Na-K-ATPase both in expression systems and in tissues, 2) distinct modulation of Na-K-ATPase activity by different FXYD proteins, 3) Na-K-ATPase isozyme-specific association with and/or modulation by different FXYD proteins, and 4) FXYD protein-deficient mice. Below is a summary of the most recent studies that support that FXYD proteins are auxiliary subunits of Na-K-ATPase and deals with the characterization of each FXYD protein (some relevant structural and functional properties of FXYD proteins are summarized in Table 1). (For further reference, see Refs. 18, 20, 92.)
FXYD1 (PHOSPHOLEMMAN)
FXYD1 is mainly expressed in the heart, skeletal muscle, and liver ( 10, 71). Detection of FXYD1 in the juxtaglomerular apparatus of the kidney indicates that FXYD1 can also be expressed in particular regions of other tissues ( 99). Expression of FXYD1 in X. laevis oocytes ( 63) or addition of FXYD1 to planar bilayers ( 17, 62) induces a Cl –-selective conductance that is activated by hyperpolarizing voltages. Moreover, FXYD1 selectively transports the zwitterionic amino acid taurine ( 62), an osmolyte of animal cells. In response to cell swelling, taurine efflux and the regulatory decrease in cell volume increase in FXYD1-transfected cells ( 64). Finally, taurine efflux in astrocytes is inhibited after a decrease in endogenous FXYD1 by antisense oligonucleotides ( 65). Thus, based on these observations, it is believed that FXYD1 plays a specific role in muscle contractility and cell volume regulation.
The recent experimental evidence that FXYD1 interacts with Na-K-ATPase adds a new perspective to the potential physiological role of FXYD1. FXYD1 interacts specifically with 1-isozymes in native cardiac and skeletal muscle ( 19, 35, 85) and with 1, 2, and 3 isoforms in the cerebellum and choroid plexus ( 32). After expression in X. laevis oocytes, FXYD1 decreases the apparent Na + affinity and, to a lesser extent, the apparent K + affinity of Na-K-ATPase ( 19). In contractile tissues, the existence of low-Na +-affinity Na/K pumps may be necessary to permit efficient extrusion of increased intracellular Na + during action potentials and thus to control appropriate muscle contractility. Alternatively, the existence of a low-Na +-affinity Na/K pump associated with FXYD1 could lead to increased intracellular Na + concentrations, an increase in intracellular Ca 2+ concentrations due to inhibition of Ca 2+ efflux via the Na/Ca exchanger, and ultimately an increased contractility of heart and skeletal muscles. However, this hypothesis is not supported by recent data, which show that at high extracellular Ca 2+ concentrations, FXYD1 overexpression in rat myocytes leads to a reduction ( 102) and downregulation of FXYD1 and thus to increased ( 60) contractility. Overexpression of FXYD1 was correlated with inhibition of the Na/Ca exchanger of both its forward and reverse exchange activity ( 102), and downregulation with an enhancement of Na/Ca exchanger function ( 60). FXYD1 colocalizes with the Na/Ca exchanger, and coimmunoprecipitation experiments suggest a direct interaction between the two proteins ( 60), indicating that FXYD1 associates not only with Na-K-ATPase but also with the Na/Ca exchanger.
The physiological role of FXYD1 has been investigated in generations of FXYD1-deficient mice ( 45). These mice show an increased ejection fraction, an increased cardiac mass in the absence of hypertension, and a significant reduction in intrinsic Na-K-ATPase activity with little decrease in total Na-K- ATPase expression. These results were interpreted to indicate that FXYD1 modulates the Na-K-ATPase and that abolishment of FXYD1 expression reduces Na-K-ATPase activity, which leads to compensatory responses. However, because the Na-K-ATPase, Na/Ca exchanger, and FXYD1 are likely to colocalize in the transverse tubules of cardiomyocytes ( 61) and Na-K-ATPase 1- and 2-isozymes interact with the Na/Ca exchanger ( 28), the relative importance of the Na-K-ATPase or the Na/Ca exchanger, or perhaps other interacting proteins, in the functional effects in FXYD1-deficient mice remains to be elucidated. Moreover, the mechanism by which FXYD1 influences Na-K-ATPase in situ in native tissues is still unclear. The results obtained with FXYD1-deficient mice ( 45) suggest that FXYD1 stimulates rather than inhibits Na-K-ATPase, and no difference was seen in the apparent Na + affinity of the Na-K-ATPase in wild-type and FXYD1-deficient mice in contrast to the previously described decrease in the Na + affinity of Na-K-ATPase induced by mammalian FXYD1 ( 19) or by the phospholemman-like shark protein ( 55).
Phosphorylation may be at least one factor that regulates the putative multiple functions of FXYD1. Considered as the major substrate for protein kinase A in the heart ( 71), FXYD1 is phosphorylated on specific serine residues by PKA and PKC and the protein kinase never in mitosis A (NIMA) ( 53, 69, 96). PKA but not PKC phosphorylation of FXYD1 increases the FXYD1-mediated currents and its cell surface expression, whereas similar effects by NIMA kinases are independent of FXYD1 phosphorylation ( 69). On the other hand, phosphorylation by myotonic dystrophy kinase induces degradation of FXYD1 ( 68). Moreover, it has been suggested that PKC phosphorylation of a phospholemman-like protein from shark induces its dissociation from Na-K-ATPase and an increase in the Na + affinity of Na-K-ATPase ( 54). In contrast, in isolated sarcolemma, ischemia produces a substantial activation of Na-K-ATPase that was correlated with PKA phosphorylation of FXYD1 without influencing the association efficiency of FXYD1 with Na-K-ATPase ( 35). Finally, PKA phosphorylation of serine 68 in FXYD1 was implicated in the increased Na-K-ATPase pump currents measured in forskolin-treated ventricular myocytes ( 85).
In conclusion, compelling evidence suggests that FXYD1 is an auxiliary subunit of Na-K-ATPase in contractile tissues and in the brain. So far, the intrinsic physiological role in these tissues is not known. In the heart and skeletal muscle, Na-K-ATPase is tightly linked to contractility, and regulation of its kinetic properties by FXYD1 may have distinct effects on this tissue function. Because both 1- and 2-isozymes are implicated in cardiac contractility ( 28), the specific association of FXYD1 with 1-isozymes suggests a subtle difference in the mechanism underlying the function of 1-isozymes. The apparently discrepant results obtained regarding the mechanism of action of the regulatory effect of FXYD1 on Na-K-ATPase activity may partly be due to variations in its phosphorylation but could also be a result of an interplay between different functional roles of FXYD1, e.g., interaction with Na/Ca exchanger and channel formation.
FXYD2 (-SUBUNIT OF Na-K-ATPase)
The FXYD2 gene is located on chromosome 11q23 ( 87). It encodes two splice variants, FXYD2a and FXYD2b (a and b), which have been identified by mass spectroscopy and differ only in their most NH 2-terminal amino acids ( 49). FXYD2 is predominantly expressed in the kidney ( 59, 90) with segment specific distribution of FXYD2a and FXYD2b ( 4, 73) ( Table 2). FXYD2a and FXYD2b colocalize with Na-K- ATPase in the basolateral membrane of renal epithelial cells. No FXYD2 was detected in the collecting duct ( 4, 73).
FXYD2 was the first FXYD protein that was found to be associated with Na-K-ATPase ( 33, 59) and to produce a functional effect on its transport properties ( 8, 90). In contrast to FXYD1, no other functional role than that of a Na-K-ATPase regulator has been proposed, although it was reported that FXYD2 expressed in X. laevis oocytes activates Ca 2+- and voltage-gated, nonselective pores endogenous to the oocyte ( 80). Evidence provided from different experimental approaches suggests that FXYD2 may have several concomitant and independent effects on Na-K-ATPase function. FXYD2 was shown to increase the apparent K + affinity of Na-K-ATPase at high negative membrane potentials in both the presence and absence of extracellular Na + ( 6). On the other hand, FXYD2 decreases the apparent K + affinity at less negative membrane potentials but only in the presence of extracellular Na +, suggesting a shift in E1-E2 equilibrium toward the E1 conformation ( 6). FXYD2 also increases the affinity for ATP ( 90, 91), which is consistent with a shift toward the E1 conformation. Moreover, it has been reported that FXYD2 increases the K + antagonism of intracellular Na + binding, suggesting an additional effect of FXYD2 on intrinsic binding of K + at cytoplasmic sites ( 73). Finally, FXYD2 decreases the Na + activation of Na/K pump currents ( 6) and produces a parallel decrease in the Na + and K + activation of Na-K-ATPase activity ( 3).
Intriguingly, the two splice variants, FXYD2a and FXYD2b, produce identical effects on the catalytic and transport properties of Na-K-ATPase studied so far ( 6, 73). However, the functional effects of FXYD2a and FXYD2b differ depending on nonidentified posttranslational modifications, which may be cell specific or depend on the physiological state ( 2). Posttranslational modifications abolish the effect of FXYD2a on the apparent Na + affinity of Na-K-ATPase activity. On the other hand, modifications of FXYD2b do not influence the effect on the apparent Na + affinity, whereas modifications are needed for the effect of FXYD2b on the apparent K + affinity ( 2). Finally, FXYD2a, but not FXYD2b, is enriched in caveolae of renal membranes ( 31), providing another argument for different functional roles of FXYD2 variants.
The physiological relevance of Na-K-ATPase modulation by FXYD2 in the kidney remains speculative. FXYD2 are mainly distributed in renal segments, which reabsorb most of the filtered Na + load. Because a major effect of FXYD2 appears to be a decrease in the Na + affinity of Na-K-ATPase, it may be speculated that the existence of low-Na +-affinity Na-K- ATPase may be favorable for an efficient reabsorption of Na + in renal segments with high cellular Na + load ( 6). The physiological importance of the modulation of the Na + affinity of Na-K-ATPase by FXYD2 is also supported by the observation that FXYD2 transfectants in culture, which reduce Na + affinity, decrease cellular growth ( 2). On the other hand, in view of the increased affinity for ATP of Na-K-ATPase associated with FXYD2, it has been suggested that FXYD2 may be important in preserving the Na-K-ATPase activity in renal segments such as the outer medulla, which are highly prone to anoxia ( 73, 91). Significantly, renal Na-K-ATPase of FXYD2-deficient mice exhibits an increased apparent Na + affinity ( 46), confirming the relevance of the Na + effect of FXYD2 observed in different expression systems. On the other hand, mice lacking FXYD2 show no obvious defects of renal function, suggesting compensatory mechanisms that permit a normal Na + excretion balance ( 46).
Mutations in a conserved glycine residue in the TM of FXYD2 have been linked to cases of human primary hypomagnesemia ( 58). Although studies on the effect of the G/R mutation have revealed that it abolishes association of FXYD proteins with the Na-K-ATPase without a change in the cell surface expression of the Na-K-ATPase ( 23, 74), further investigations are needed to elucidate whether or how this effect may be associated with the loss of Mg 2+, increased Ca 2+ absorption, and hypocalciuria observed in these patients.
Regulation of FXYD2 expression has been studied during development and in the adult kidney. During amphibian development, FXYD2 is found in several tissues but mainly in the developing pronephric kidney ( 29). Expression of FXYD2 in the kidney coincides with the onset of expression of Na-K-ATPase 1- and 1-subunits, suggesting common regulatory mechanisms. This pattern of expression highlights the functional importance of FXYD2 in the modulation of Na-K-ATPase. In cultured murine inner medullary collecting duct (IMCD3) cells lacking FXYD2, the expression of both FXYD2a and FXYD2b is induced by hypertonicity depending on c-Jun kinase and phosphatidylinositol 3-kinase activation ( 12). Upregulated FXYD2 is routed to the basolateral membrane of IMCD3 cells, consistent with its localization in inner medullary collecting duct cells in vivo ( 72). The osmotic regulation of FXYD2 is also observed in mouse inner renal medulla in response to changes in hydration ( 12). In IMCD3 cells, failure to synthesize FXYD2 in response to hypertonicity leads to decreased cell viability, suggesting a potential role of the regulation of Na-K-ATPase by FXYD2 in cell survival under anisotonic conditions ( 12). Upregulation of FXYD2 and c-Jun kinase activation during hypertonicity is mediated by chloride but not by sodium, in contrast to upregulation of the Na-K-ATPase -subunit induced by sodium ( 14). In IMCD3 cells, activation of c-Jun kinase regulates FXYD2 at the transcriptional level, whereas activation of PI3-kinase influences the translation of FXYD2 ( 13). Finally, FXYD2a but not FXYD2b expression is induced in renal cells of proximal origin as well as in several cell lines of other than renal origin by hypertonicity, heat shock, and chemical stress ( 98). Moreover, injury-induced NF-B activation induces FXYD2 expression in the hippocampus ( 48). FXYD2a induction in cells of renal origin is accompanied by a reduction in Na-K-ATPase activity and in the rate of cell division ( 98). Both of these effects are reduced when FXYD2 expression was knocked down by small interfering RNA (siRNA), suggesting that induction of FXYD2a may be part of a cellular response to genotoxic stress ( 98).
Although much has been learned about the functional effects of FXYD2 on Na-K-ATPase and on its regulation, we are far from understanding the physiological relevance of this protein and its potential role in pathophysiological states such as human primary hypomagnesemia.
FXYD3 (Mat-8)
FXYD3 has initially been identified in murine breast tumors induced by Neu or Ras oncogenes ( 66) and was then also found to be present in primary human breast tumors and in human breast tumor cell lines ( 67), in colorectal cancer cell lines ( 101), and in prostate tumors ( 41). In normal tissue, FXYD3 is mainly expressed in the uterus, stomach, colon, and skin ( 67) ( 77). In adult mice, FXYD3 and Lgi4 genes have been mapped to the same locus on chromosome 7 and produce partially overlapping transcripts of opposite orientation, which potentially may impact on their respective expression ( 77).
When expressed in X. laevis oocytes, FXYD3 induces a hyperpolarization-activated chloride conductance similar to that observed with FXYD1 ( 67). Moreover, FXYD3 can associate with Na-K-ATPase and produces a decrease in both the apparent K + and Na + affinity of Na-K-ATPase ( 22). Interestingly, in addition to these functions shared with other FXYD proteins, FXYD3 exhibits some uncommon characteristics. First, in contrast to other FXYD proteins, FXYD3 may have two TMs because its signal sequence is not cleaved. Second, when expressed in X. laevis oocytes, FXYD3 can associate not only with Na-K-ATPase but also with gastric and colonic H-K-ATPase. However, in situ (stomach), FXYD3 is only expressed in mucous cells, which lack H-K-ATPase. Finally, after expression in X. laevis oocytes, FXYD3 modulates the processing of glycoproteins ( 22).
At present, it is difficult to draw conclusions about the physiological relevance of the Na + and K + effects of FXYD3 on Na-K-ATPase. FXYD3 is highly upregulated in specific types of cancers, such as breast or prostate tumors. Interestingly, FXYD3 has been shown to be upregulated by 5-Fluorouracil treatment of breast cancer cells in a p53-dependent way ( 57). Because p53 is an antiproliferative protein, one may assume that FXYD3 could be part of this function. However, Grzmil et al. ( 41) showed that siRNA-mediated inhibition of FXYD3 expression causes a significant reduction of cell proliferation of prostate cancer cell lines, indicating instead that FXYD3 could be involved in cell proliferation. Possibly, p53 may induce genes with proliferative functions as part of a negative feedback mechanism. It remains to be shown whether regulation of the functional properties of Na-K-ATPase, leading to modifications of the cellular milieu, is important in impeding or triggering cell proliferation. Finally, because FXYD3 not only colocalizes with Na-K-ATPase in basolateral membranes but is also found in apical membranes of mucous cells of the stomach ( 22), it cannot be excluded that FXYD3 is not only a Na-K-ATPase modulator but also has other yet unknown functions.
FXYD4 (CHIF)
FXYD4 is expressed in kidney medullary collecting duct and papilla and in the distal colon ( 16, 81) ( Table 2). Like FXYD2, FXYD4 is only expressed in basolateral membranes of target epithelia.
Because FXYD4 induces a depolarization-dependent K +-specific ion conductance when expressed in X. laevis oocytes, it has been suggested that FXYD4 may be a regulator of ion channels ( 5). However, this channel activity turned out to be irreproducible ( 81), and it is more likely that, as suggested by more recent evidence, FXYD4 associates specifically with Na-K-ATPase in expression systems and in situ, as well as modulates its transport properties ( 6, 37).
In the absence of extracellular Na +, a situation in which K + kinetics most closely reflect the intrinsic affinity for extracellular K +, FXYD4 has no effect on the K + activation of Na-K-ATPase. On the other hand, in the presence of extracellular Na +, a condition in which the K + kinetics depend on the competition by extracellular Na +, FXYD4 produces a large increase in the K1/2 value for K + of the Na-K-ATPase ( 6). Moreover, FXYD4 induces a two- to threefold increase in the apparent affinity for intracellular Na + ( 6, 37). This Na + effect of FXYD4 on Na-K-ATPase is likely to be of physiological relevance for the Na + reabsorption process in the collecting duct, which is the ultimate site of electrolyte conservation. An increase in the Na + affinity of Na-K-ATPase produced by the association with FXYD4 in this renal segment is favorable because it permits efficient Na + reabsorption even at low intracellular Na + concentrations. At physiologically low intracellular Na + concentrations (e.g., 5 mM), the Na-K-ATPase transport rate of Na-K-ATPase associated with FXYD4 should be about four times higher than that of Na-K-ATPase lacking FXYD4 ( 6).
Results from FXYD4 knockout mice ( 1, 40) at least partially confirm that a major if not unique effect of FXYD4 is the activation of Na-K-ATPase by increasing its apparent Na + affinity. Abolition of FXYD4 expression appears to be fully compensated for in the kidney because fractional excretions of Na + and K + are normal under resting conditions, and animals have no deficit in the adaptation to low Na + and K + intake. However, in the distal colon, amiloride-inhibitable Na + reabsorption is reduced under control conditions, glucocorticoid treatment, and low Na + intake ( 40).
Regulation of FXYD4 expression in the kidney and colon is different. Both distal colon and the renal collecting duct, where FXYD4 is expressed, are aldosterone target sites, but aldosterone induces FXYD4 cRNA expression only in the colon but not in the kidney ( 11, 97). On the other hand, low Na + intake increases FXYD4 mRNA and protein expression in the colon, but only FXYD4 protein and not mRNA expression in the kidney ( 16, 81). Moreover, in experimental ischemic acute renal failure ( 39, 75) and in acute tubular necrosis ( 84), which are characterized by hyperkalemia, FXYD4 cRNA expression is decreased in kidney and increased in colon. Possibly, suppression of FXYD4 expression in kidney could lead to decreased renal K + secretion, and the enhanced FXYD4 expression in colon might be an adaptive response.
Although compelling evidence suggests that an important biological activity of FXYD4 is mediated through its interaction with Na-K-ATPase, it remains open whether this is the only function of FXYD4 or whether FXYD4 interacts with and regulates other, as yet unidentified, partner proteins.
FXYD5 (Ric) AND FXYD6 (PHOSPHOHIPPOLIN)
FXYD5 ( 34) and FXYD6 ( 100) are poorly characterized, and interaction with and modulation of Na-K-ATPase have so far not been reported.
Compared with other FXYD proteins, FXYD5 has an unusual long NH 2-terminal extension ( 86). The so-called IUW-1 protein, which may be an isoform of FXYD5, has a shorter NH 2 terminus and shows 61% sequence identity with the cytoplasmic domain of the angiotensin II type 1 receptor ( 70). FXYD5 expression is induced in cells transformed by the oncogene E2a-Pbxl ( 34). FXYD5 is also expressed in several cancer tissues but only in a few normal cell types ( 44). A role of FXYD5 in tumor progression and metastasis has been suggested, based on the observations that transfection of FXYD5, called dysadherin, into liver cancer cells results in decreased E-cadherin-mediated cell-cell adhesion ( 44), implicating O-glycosylation of FXYD5 ( 94). Moreover, dysadherin positivity was correlated with poor prognosis in various human cancers ( 82, 83).
FXYD6 is expressed in several tissues ( 86). In the brain, expression studies during development suggest a role in neuronal excitability during postnatal development and in the adult brain ( 47, 78).
FXYD7
FXYD7 has been found exclusively in the brain, where it is expressed in neurons and to a lesser extent in glial cells ( 7). FXYD7 is subjected to O-glycosylation, which appears to be necessary for the stability of the protein ( 7).
Mutational analysis of FXYD7-specific regions revealed a COOH-terminal, cytoplasmic valine residue that is involved in rapid endoplasmic reticulum export and controls the rate of its cell surface expression ( 23). After coexpression in X. laevis oocytes, FXYD7 associates posttranslationally with Na-K-ATPase ( 23), probably due to the different rates of endoplasmic reticulular exit of the two proteins. Association of FXYD7 occurs with 1 1, 2 1, and 3 1 isozymes but not with Na-K-ATPase isozymes containing 2-isoforms ( 7). On the other hand, in the brain, FXYD7 exclusively associates with Na-K-ATPase containing 1-isoforms ( 7). These data suggest that in the brain, FXYD7 is specifically associated with Na-K-ATPase 1 1-isozymes.
Electrophysiological analysis of the modulatory effect of FXYD7 on Na-K-ATPase in X. laevis oocytes revealed that FXYD7 modulates the transport properties of 1 1-isozymes in a specific way, distinct from other FXYD proteins ( 7). The apparent affinity for K + is increased over a wide range of membrane potentials in both the presence and absence of extracellular Na +, which suggests a modification of the intrinsic affinity of the external K +-binding site. On the other hand, FXYD7 does not influence the apparent affinity for intracellular Na + ( 7). It can be speculated that in the brain, the existence of Na-K-ATPase 1 1-isozymes with low K + affinity, acquired by association with FXYD7, may be necessary for efficient clearance of extracellular K + during neuronal activity to ensure neuronal excitability.
Future studies should be directed to determine whether the endoplasmic reticular export control of FXYD7 dependent on the COOH-terminal valine residue may be linked to the specific expression of FXYD7 in neurons and glial cells and/or to the particular requirements of the regulation of Na-K-ATPase expression and function in the brain.
STRUCTURE-FUNCTION RELATIONSHIP
In addition to the elucidation of the functional effects on Na-K-ATPase of several FXYD proteins, recent studies have revealed details on the molecular basis of these effects and on the structural and functional interaction sites in Na-K-ATPase and FXYD proteins. It is becoming clear that multiple sites of interaction exist that involve both the transmembrane and extramembrane domains.
The role of conserved amino acids in the structural and functional interaction with Na-K-ATPase has been studied in some FXYD proteins. Replacement of Gly-41 with Arg in the TM domain of FXYD2 ( 103), which is associated with a form of renal hypomagnesemia ( 58), or replacement of the analogous Gly-40 with Arg in FXYD7 ( 23) abolishes the interaction of these FXYD proteins with Na-K-ATPase. Replacements with Leu of Gly-41 in FXYD2 ( 103) or with Ala or Trp of Gly-40 in FXYD7 ( 23) permit partial association with Na-K-ATPase, which is paralleled by a partial loss of the effect of FXYD7 on the apparent K + affinity of Na-K-ATPase ( 23). A similar reduction of the association efficiency and the K + effect is also observed when the other conserved Gly-29 in FXYD7 is replaced by Ala. Gly-40 and Gly-29 are on the same side of the TM -helix of FXYD proteins and form a groove that could be important as an interaction site between Na-K-ATPase and FXYD proteins. On the other hand, experiments using synthetic transmembrane mimetic peptides suggest that Gly-41 in FXYD2 is important for the effect of FXYD2 on the apparent Na + affinity of Na-K-ATPase and not primarily on the association of FXYD2 with Na-K-ATPase ( 103). Finally, experiments using perfluorooctanoate gel electrophoresis indicate that FXYD2 and FXYD1 ( 56) and peptides corresponding to the TM domain of FXYD2 ( 89) may form oligomers. Gly-41 is necessary for oligomerization of FXYD2 peptides ( 89).
The role of the conserved FXYD motif has been studied in several FXYD proteins, but so far no preserved function could be revealed. The FXYD motif is important for the stable interaction of Na-K-ATPase with FXYD2 and FXYD4 ( 6) but not with FXYD7 ( 23).
Because an antibody against the COOH terminus of FXYD2 ( 73, 91) and truncations of the COOH and the NH 2 termini of FXYD2 ( 74) abolish the effect of FXYD2 on the apparent affinity for ATP of Na-K-ATPase, it was concluded that extramembrane interactions may be important for this functional effect of FXYD2. Interestingly, in FXYD2-deficient mice, the ATP affinity of Na-K-ATPase was not decreased as expected, but slightly increased, suggesting that extramembrane interactions may be context specific ( 46). On the other hand, by examining FXYD2/FXYD4 chimera, it was found that TM interactions of FXYD proteins determine both the stability of FXYD proteins in detergents and the effects of these FXYD proteins on the Na + affinity of Na-K-ATPase ( 52). Interestingly, different amino acids in the TM appear to mediate the stability and the functional effects of these FXYD proteins. The functional role of TM interactions in the Na + effect of FXYD2 was confirmed by showing that peptides corresponding to the TM domain of FXYD2 decrease the Na + affinity of Na-K-ATPase similarly to full-length FXYD2 ( 103).
Recent experimental evidence also shed some light on the sites where FXYD proteins interact with Na-K-ATPase / complexes. Based on experiments using thermal denaturation, it has been suggested that FXYD2 interacts with TM8–10 of the Na-K-ATPase -subunit ( 27). Moreover, electron crystallographic analysis of renal Na-K-ATPase at 9.5- resolution ( 42) and taking as a basis the high-resolution structure of the Ca-ATPase ( 93) suggest that FXYD2 is located in a binding pocket made up of TM9, TM6, TM4, and TM2. A role of TM9 in the structural and functional interaction with FXYD proteins could be confirmed by mutational analysis ( 51). Interestingly, Leu 964 and Phe 967 of TM9 of the rat Na-K-ATPase contribute to the stable interaction with FXYD2, FXYD4, and FXYD7 but do not influence the functional effect of these FXYD proteins on the apparent K + affinity of Na-K-ATPase. On the other hand, Phe 956 and Glu 960 do not contribute to the efficient association of FXYD proteins but transmit the K + effect of FXYD proteins to Na-K-ATPase. The relative contribution of Phe 956 and Glu 960 in the K + effect of different FXYD proteins is different, probably reflecting the intrinsic variations in the modulation of the apparent K + affinity of Na-K-ATPase by different FXYD proteins. In contrast to the K + effect, the modulation of the apparent Na + affinity of Na-K-ATPase by FXYD4 is not mediated by Phe 956 and Glu 960. The mutational analysis is in good agreement with a docking model of the Na-K-ATPase -subunit/FXYD7 complex ( 51). Thus the structural and functional interaction with FXYD proteins involve TM9 of the Na-K-ATPase -subunit but probably also other TM domains that remain to be determined. Based on cross-linking data, interactions of FXYD2 with the intracellular loop L6/7 in the Na-K-ATPase -subunit and with the extracellular domain of the Na-K-ATPase -subunit can also be predicted ( 36). Moreover, modeling of Na-K-ATPase/FXYD2 complexes is in agreement with the position of FXYD2 in a binding pocket composed of TM9, TM6, TM4, and TM2.
In conclusion, recent experimental evidence strongly supports that at least one functional role of FXYD proteins is the regulation of Na-K-ATPase in a tissue- and isoform-specific way. Although the functional effects of FXYD proteins on the transport and kinetic properties of Na-K-ATPase have been well characterized, much still remains to be learned about the physiological relevance of these effects and the potential implication of a loss of FXYD regulation of Na-K-ATPase in pathophysiological states. For a better understanding of these issues, more studies are needed on the functional roles of each FXYD protein, on their biosynthesis and processing, their tissue and cellular distribution, and their structure-function relationship.
GRANTS
This work was supported by Swiss National Fund Grant 3100A0–107513/1 (to K. Geering).
FOOTNOTES
REFERENCES
Aizman R, Asher C, Fuzesi M, Latter H, Lonai P, Karlish SJD, and Garty H. Generation and phenotypic analysis of CHIF knockout mice. Am J Physiol Renal Physiol 283: F569–F577, 2002.
Arystarkhova E, Donnet C, Asinovski NK, and Sweadner KJ. Differential regulation of renal Na,K-ATPase by splice variants of the subunit. J Biol Chem 277: 10162–10172, 2002.
Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. The subunit modulates Na + and K + affinity of the renal Na,K-ATPase. J Biol Chem 274: 33183–33185, 1999.
Arystarkhova E, Wetzel RK, and Sweadner KJ. Distribution and oligomeric association of splice forms of Na +-K +-ATPase regulatory -subunit in rat kidney. Am J Physiol Renal Physiol 282: F393–F407, 2002.
Attali B, Latter H, Rachamim N, and Garty H. A corticosteroid-induced gene expressing an "IsK-like" K + channel activity in Xenopus oocytes. Proc Natl Acad Sci USA 92: 6092–6096, 1995.
Beguin P, Crambert G, Guennoun S, Garty H, Horisberger JD, and Geering K. CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the -subunit. EMBO J 20: 3993–4002, 2001.
Beguin P, Crambert G, Monnet-Tschudi F, Uldry M, Horisberger JD, Garty H, and Geering K. FXYD7 is a brain-specific regulator of Na,K-ATPase 1- isozymes. EMBO J 21: 3264–3273, 2002.
Beguin P, Wang XY, Firsov D, Puoti A, Claeys D, Horisberger JD, and Geering K. The subunit is a specific component of the Na,K-ATPase and modulates its transport properties. EMBO J 16: 4250–4260, 1997.
Blanco G and Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 275: F633–F650, 1998.
Bogaev RC, Jia LG, Kobayashi YM, Palmer CJ, Mounsey JP, Moorman JR, Jones LR, and Tucker AL. Gene structure and expression of phospholemman in mouse. Gene 271: 69–79, 2001.
Brennan FE and Fuller PJ. Acute regulation by corticosteroids of channel-inducing factor gene messenger ribonucleic acid in the distal colon. Endocrinology 140: 1213–1218, 1999.
Capasso JM, Rivard C, and Berl T. The expression of the subunit of Na-K-ATPase is regulated by osmolality via C-terminal Jun kinase and phosphatidylinositol 3-kinase-dependent mechanisms. Proc Natl Acad Sci USA 98: 13414–13419, 2001.
Capasso JM, Rivard CJ, and Berl T. Synthesis of the Na-K-ATPase -subunit is regulated at both the transcriptional and translational levels in IMCD3 cells. Am J Physiol Renal Physiol 288: F76–F81, 2005.
Capasso JM, Rivard CJ, Enomoto LM, and Berl T. Chloride, not sodium, stimulates expression of the subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells. Proc Natl Acad Sci USA 100: 6428–6433, 2003.
Capendeguy O and Horisberger JD. Functional effects of Na +,K +-ATPase gene mutations. Neuromol Med 6: 105–116, 2004.
Capurro C, Coutry N, Bonvalet JP, Escoubet B, Garty H, and Farman N. Cellular localization and regulation of CHIF in kidney and colon. Am J Physiol Cell Physiol 271: C753–C762, 1996.
Chen Z, Jones LR, O'Brian JJ, Moorman JR, and Cala SE. Structural domains in phospholemman: a possible role for the carboxyl terminus in channel inactivation. Circ Res 82: 367–374, 1998.
Cornelius F and Mahmmoud YA. Functional modulation of the sodium pump: the regulatory proteins "Fixit." News Physiol Sci 18: 119–124, 2003.
Crambert G, Fuzesi M, Garty H, Karlish S, and Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci USA 99: 11476–11481, 2002.
Crambert G and Geering K. FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. Sci STKE 166: RE1, 2003.
Crambert G, Hasler U, Beggah AT, Yu C, Modyanov NN, Horisberger JD, Lelievre L, and Geering K. Transport and pharmacological properties of nine different human Na,K-ATPase isozymes. J Biol Chem 275: 1976–1986, 2000.
Crambert G, Li C, Claeys D, and Geering K. FXYD3 (Mat-8), a new regulator of Na,K-ATPase. Mol Biol Cell 16: 2363–2371, 2005.
Crambert G, Li C, Swee LK, and Geering K. FXYD7: mapping of functional sites involved in endoplasmic reticulum export, association with and regulation of Na,K-ATPase. J Biol Chem 279: 30888–30895, 2004.
Crowell KJ, Franzin CM, Koltay A, Lee S, Lucchese AM, Snyder BC, and Marassi FM. Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers. Biochim Biophys Acta 1645: 15–21, 2003.
De Carvalho Aguiar P, Sweadner KJ, Penniston JT, Zaremba J, Liu L, Caton M, Linazasoro G, Borg M, Tijssen MA, Bressman SB, Dobyns WB, Brashear A, and Ozelius LJ. Mutations in the Na +/K +-ATPase 3 gene ATP1A3 are associated with rapid-onset dystonia parkinsonism. Neuron 43: 169–175, 2004.
De Fusco M, Marconi R, Silvestri L, Atorino L, Rampoldi L, Morgante L, Ballabio A, Aridon P, and Casari G. Haploinsufficiency of ATP1A2 encoding the Na +/K + pump 2 subunit associated with familial hemiplegic migraine type 2. Nat Genet 33: 192–196, 2003.
Donnet C, Arystarkhova E, and Sweadner KJ. Thermal denaturation of the Na,K-ATPase provides evidence for - oligomeric interaction and subunit association with the C-terminal domain. J Biol Chem 276: 7357–7365, 2001.
Dostanic I, Schultz JEJ, Lorenz JN, and Lingrel JB. The 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart. J Biol Chem 279: 54053–54061, 2004.
Eid SR and Brandli AW. Xenopus Na,K-ATPase: primary sequence of the 2 subunit and in situ localization of 1, 1, and expression during pronephric kidney development. Differentiation 68: 115–125, 2001.
Feraille E and Doucet A. Sodium-potassium-adenosine triphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345–418, 2001.
Ferrandi M, Molinari I, Barassi P, Minotti E, Bianchi G, and Ferrari P. Organ hypertrophic signaling within caveolae membrane subdomains triggered by ouabain and antagonized by PST 2238. J Biol Chem 279: 33306–33314, 2004.
Feschenko MS, Donnet C, Wetzel RK, Asinovski NK, Jones LR, and Sweadner KJ. Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus. J Neurosci 23: 2161–2169, 2003.
Forbush B III, Kaplan JH, and Hoffman JF. Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na,K-ATPase. Biochemistry 17: 3667–3676, 1978.
Fu X and Kamps M. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts. Mol Cell Biol 17: 1503–1512, 1997.
Fuller W, Eaton P, Bell JR, and Shattock MJ. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K ATPase. FASEB J 18: 197–199, 2003.
Fuzesi M, Gottschalk KE, Lindzen M, Shainskaya A, Kuster B, Garty H, and Karlish SJ. Covalent cross-links between the subunit(FXYD2) and and subunits of Na,K-ATPase. Modeling the - interaction. J Biol Chem 280: 18291–18301, 2005.
Garty H, Lindzen M, Scanzano R, Aizman R, Fuzesi M, Goldshleger R, Farman N, Blostein R, and Karlish SJD. A functional interaction between CHIF and Na-K-ATPase: implication for regulation by FXYD proteins. Am J Physiol Renal Physiol 283: F607–F615, 2002.
Geering K. The functional role of subunits in oligomeric P-type ATPases. J Bioenerg Biomembr 33: 425–438, 2001.
Gimelreich D, Popovtzer MM, Wald H, Pizov G, Berlatzky Y, and Rubinger D. Regulation of ROMK and channel-inducing factor (CHIF) in acute renal failure due to ischemic reperfusion injury. Kidney Int 59: 1812–1820, 2001.
Goldschimdt I, Grahammer F, Warth R, Schulz-Baldes A, Garty H, Greger R, and Bleich M. Kidney and colon electrolyte transport in CHIF knockout mice. Cell Physiol Biochem 14: 113–120, 2004.
Grzmil M, Voigt S, Thelen P, Hemmerlein B, Helmke K, and Burfeind P. Up-regulated expression of the MAT-8 gene in prostate cancer and its siRNA-mediated inhibition of expression induces a decrease in proliferation of human prostate carcinoma cells. Int J Oncol 24: 97–105, 2004.
Hebert H, Purhonen P, Vorum H, Thomsen K, and Maunsbach AB. Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals. J Mol Biol 314: 479–494, 2001.
Horisberger JD. Recent insights into the structure and mechanism of the sodium pump. Physiology (Bethesda) 19: 377–387, 2004.
Ino Y, Gotoh M, Sakamoto M, Tsukagoshi K, and Hirohashi S. Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis. Proc Natl Acad Sci USA 99: 365–370, 2002.
Jia LG, Donnet C, Bogaev RC, Blatt RJ, McKinney CE, Day KH, Berr SS, Jones LR, Moorman JR, Sweadner KJ, and Tucker AL. Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice. Am J Physiol Heart Circ Physiol 288: H1982–H1988, 2005.
Jones DH, Li TY, Arystarkhova E, Barr KJ, Wetzel RK, Peng J, Markham K, Sweadner KJ, Fong GH, and Kidder GM. Na,K-ATPase from mice lacking the subunit (FXYD2) exhibits altered Na + affinity and decreased thermal stability. J Biol Chem 280: 19003–19011, 2005.
Kadowaki K, Sugimoto K, Yamaguchi F, Song T, Watanabe Y, Singh K, and Tokuda M. Phosphohippolin expression in the rat central nervous system. Mol Brain Res 125: 105–112, 2004.
Kassed CA, Butler TL, Patton GW, Demesquita DD, Navidomskis MT, Memet S, Israel A, and Pennypacker KR. Injury-induced NF-B activation in the hippocampus: implications for neuronal survival. FASEB J 18: 723–724, 2004.
Kuster B, Shainskaya A, Pu HX, Goldshleger R, Blostein R, Mann M, and Karlish SJ. A new variant of the subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells. J Biol Chem 275: 18441–18446, 2000.
Laski ME and Kurtzman NA. The renal adenosine triphosphatases—functional integration and clinical significance. Miner Electrolyte Metab 22: 410–422, 1996.
Li C, Grosdidier A, Crambert G, Horisberger JD, Michielin O, and Geering K. Structural and functional interaction sites between Na,K-ATPase and FXYD proteins. J Biol Chem 279: 38895–38902, 2004.
Lindzen M, Aizman R, Lifshitz Y, Lubarski I, Karlish SJD, and Garty H. Structure-function relations of interactions between Na,K-ATPase, the subunit, and corticosteroid hormone-induced factor. J Biol Chem 278: 18738–18743, 2003.
Lu KP, Kemp BE, and Means AR. Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J Biol Chem 269: 6603–6607, 1994.
Mahmmoud YA and Cornelius F. Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the - and -subunits close to the inner face of the plasma membrane. Biophys J 82: 1907–1919, 2002.
Mahmmoud YA, Cramb G, Maunsbach AB, Cutler CP, Meischke L, and Cornelius F. Regulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS. J Biol Chem 278: 37427–37438, 2003.
Mahmmoud YA, Vorum H, and Cornelius F. Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase C via a novel member of the FXYDY family. J Biol Chem 275: 35969–35977, 2000.
Maxwell PJ, Longley DB, Latif T, Boyer J, Allen W, Lynch M, McDermott U, Harkin DP, Allegra CJ, and Johnston PG. Identification of 5-fluorouracil-inducible target genes using cDNA microarray profiling. Cancer Res 63: 4602–4606, 2003.
Meij IC, Koenderink JB, van Bokhoven H, Assink KF, Groenestege WT, de Pont JJ, Bindels RJ, Monnens LA, van den Heuvel LP, and Knoers NV. Dominant isolated renal magnesium loss is caused by misrouting of the Na +,K +-ATPase -subunit. Nat Genet 26: 265–266, 2000.
Mercer RW, Biemesderfer D, Bliss DP, Collins JH, and Forbush B. Molecular cloning and immunological characterization of the -polypeptide, a small protein associated with the Na,K-ATPase. J Cell Biol 121: 579–586, 1993.
Mirza MA, Zhang XQ, Ahlers BA, Qureshi A, Carl LL, Song J, Tucker AL, Mounsey JP, Moorman JR, Rothblum LI, Zhang TS, and Cheung JY. Effects of phospholemman downregulation on contractility and Ca 2+i transients in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H1322–H1330, 2004.
Mohler PJ, Schott JJ, Gramolini AO, Dilly KW, Guatimosim S, duBell WH, Song LS, Haurogne K, Kyndt F, Ali ME, Rogers TB, Lederer WJ, Escande D, Le Marec H, and Bennett V. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421: 634–639, 2003.
Moorman JR, Ackerman SJ, Kowdley GC, Griffin MP, Mounsey JP, Chen ZH, Cala SE, Obrian JJ, Szabo G, and Jones LR. Unitary anion currents trough phospholemman channel molecules. Nature 377: 737–740, 1995.
Moorman JR, Palmer CJ, John JE III, Durieux ME, and Jones LR. Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes. J Biol Chem 267: 14551–14554, 1992.
Morales-Mulia M, Pasantes-Morales H, and Moran J. Volume sensitive efflux of taurine in HEK293 cells overexpressing phospholemman. Biochim Biophys Acta 1496: 252–260, 2000.
Moran J, Morales-Mulia M, and Pasantes-Morales H. Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes. Biochim Biophys Acta 1538: 313–320, 2001.
Morrison BW and Leder P. Neu and ras initiate murine mammary tumors that share genetic markers generally absent in c-myc and int-2-initiated tumors. Oncogene 9: 3417–3426, 1994.
Morrison BW, Moorman JR, Kowdley GC, Kobayashi YM, Jones LR, and Leder P. Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. J Biol Chem 270: 2176–2182, 1995.
Mounsey JP, John JE III, Helmke SM, Bush EW, Gilbert J, Roses AD, Perryman MB, Jones LR, and Moorman JR. Phospholemman is a substrate for myotonic dystrophy protein kinase. J Biol Chem 275: 23362–2367, 2000.
Mounsey JP, Lu KP, Patel MK, Chen ZH, Horne LT, John JE, Means AR III, Jones LR, and Moorman JR. Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases. Biochim Biophys Acta 1451: 305–318, 1999.
Omasa T, Chen YG, Mantalaris A, and Wu JH. A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP11/PLM/MAT8 family of transmembrane proteins. Biochim Biophys Acta 1517: 307–310, 2001.
Palmer CJ, Scott BT, and Jones LR. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J Biol Chem 266: 11126–11130, 1991.
Pihakaski-Maunsbach K, Tokonabe S, Vorum H, Rivard CJ, Capasso JM, Berl T, and Maunsbach AB. The -subunit of Na-K-ATPase is incorporated into plasma membranes of mouse IMCD3 cells in response to hypertonicity. Am J Physiol Renal Physiol 288: F650–F657, 2005.
Pu HX, Cluzeaud F, Goldshlegger R, Karlish SJD, Farman N, and Blostein R. Functional role and immunocytochemical localization of the a and b forms of the Na,K-ATPase subunit. J Biol Chem 276: 20370–20378, 2001.
Pu HX, Scanzano R, and Blostein R. Distinct regulatory effects of the Na,K-ATPase subunit. J Biol Chem 277: 20270–20276, 2002.
Rabb H, Wang Z, Postler G, and Soleimani M. Possible molecular basis for changes in potassium handling in acute renal failure. Am J Kidney Dis 35: 871–877, 2000.
Rose AM and Valdes R. Understanding the sodium pump and its relevance to disease. Clin Chem 40: 1674–1685, 1994.
Runkel F, Michels M, and Franz T. Fxyd3 and Lgi4 expression in the adult mouse: a case of endogenous antisense expression. Mamm Genome 14: 665–672, 2003.
Saito S, Matoba R, Kato K, and Matsubara K. Expression of a novel member of the ATP11/PLM/MAT8 family, phospholemman-like protein (PLP) gene, in the developmental process of mouse cerebellum. Gene 279: 149–155, 2001.
Segall L, Scanzano R, Kaunisto MA, Wessman M, Palotie A, Gargus JJ, and Blostein R. Kinetic alterations due to a missense mutation in the Na,K-ATPase 2 subunit cause familial hemiplegic migraine type 2. J BiolChem 279: 43692–43696, 2004.
Sha Q, Lansbery KL, Distefano D, Mercer RW, and Nichols CG. Heterologous expression of the Na +,K +-ATPase subunit in Xenopus oocytes induces an endogenous, voltage-gated large diameter pore. J Physiol 535: 407–417, 2001.
Shi H, Levy-Holzman R, Cluzeaud F, Farman N, and Garty H. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505–F512, 2001.
Shimada Y, Yamasaki S, Hashimoto Y, Ito T, Kawamura J, Soma T, Ino Y, Nakanishi Y, Sakamoto M, Hirohashi S, and Imamura M. Clinical significance of dysadherin expression in gastric cancer patients. Clin Cancer Res 10: 2818–2823, 2004.
Shimamura T, Sakamoto M, Ino Y, Sato Y, Shimada K, Kosuge T, Sekihara H, and Hirohashi S. Dysadherin overexpression in pancreatic ductal adenocarcinoma reflects tumor aggressiveness: relationship to E-cadherin expression. J Clin Oncol 21: 659–667, 2003.
Shustin L, Wald H, and Popovtzer MM. Role of down-regulated CHIF mRNA in the pathophysiology of hyperkalemia of acute tubular necrosis. Am J Kidney Dis 32: 600–604, 1998.
Silverman BdZ, Fuller W, Eaton P, Deng J, Moorman JR, Cheung JY, James AF, and Shattock MJ. Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. Cardiovasc Res 65: 93–103, 2005.
Sweadner KJ and Rael E. The FXYD gene family of small ion transport regulators or channels: cDNA sequence, protein signature sequence, and expression. Genomics 68: 41–56, 2000.
Sweadner KJ, Wetzel RK, and Arystarkhova E. Genomic organization of the human FXYD2 gene encoding the of the Na,K-ATPase. Biochem Biophys Res Commun 279: 196–201, 2000.
Therien AG and Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541–C566, 2000.
Therien AG and Deber CM. Oligomerization of a peptide derived from the transmembrane region of the sodium pump subunit: effect of the pathological mutation G41R. J Mol Biol 322: 583–590, 2002.
Therien AG, Goldshleger R, Karlish SJ, and Blostein R. Tissue-specific distribution and modulatory role of the subunit of the Na,K-ATPase. J Biol Chem 272: 32628–32634, 1997.
Therien AG, Karlish SJ, and Blostein R. Expression and functional role of the subunit of the Na,K-ATPase in mammalian cells. J Biol Chem 274: 12252–12256, 1999.
Therien AG, Pu HX, Karlish SJ, and Blostein R. Molecular and functional studies of the subunit of the sodium pump. J Bioenerg Biomembr 33: 407–414, 2001.
Toyoshima C, Nakasako M, Nomura H, and Ogawa H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405: 647–655, 2000.
Tsuiji H, Takasaki S, Sakamoto M, Irimura T, and Hirohashi S. Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell-cell adhesion. Glycobiology 13: 521–527, 2003.
Vanmolkot KR, Kors EE, Hottenga JJ, Terwindt GM, Haan J, Hoefnagels WA, Black DF, Sandkuijl LA, Frants RR, Ferrari MD, and van den Maagdenberg AM. Novel mutations in the Na +,K +-ATPase pump gene ATP1A2 associated with familial hemiplegic migraine and benign familial infantile convulsions. Ann Neurol 54: 360–366, 2003.
Walaas SI, Czernik AJ, Olstad OK, Sletten K, and Walaas O. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem J 304: 635–640, 1994.
Wald H, Goldstein O, Asher C, Yagil Y, and Garty H. Aldosterone induction and epithelial distribution of CHIF. Am J Physiol Renal Fluid Electrolyte Physiol 271: F322–F329, 1996.
Wetzel RK, Pascoa JL, and Arystarkhova E. Stress-induced expression of the subunit (FXYD2) modulates Na,K-ATPase activity and cell growth. J Biol Chem 279: 41750–41757, 2004.
Wetzel RK and Sweadner KJ. Phospholemman expression in extraglomerular mesangium and afferent arteriole of the juxtaglomerular apparatus. Am J Physiol Renal Physiol 285: F121–F129, 2003.
Yamaguchi F, Yamaguchi K, Tai Y, Sugimoto K, and Tokuda M. Molecular cloning and characterization of a novel phospholemman-like protein from rat hippocampus. Brain Res Mol Brain Res 86: 189–192, 2001.
Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, and Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 96: 14517–14522, 1999.
Zhang XQ, Qureshi A, Song J, Carl LL, Tian Q, Stahl RC, Carey DJ, Rothblum LI, and Cheung JY. Phospholemman modulates Na +/Ca 2+ exchange in adult rat cardiac myocytes. Am J Physiol Heart Circ Physiol 284: H225–H233, 2003.
Zouzoulas A, Therien AG, Scanzano R, Deber CM, and Blostein R. Modulation of Na,K-ATPase by the subunit: studies with transfected cells and transmembrane mimetic peptides. J Biol Chem 278: 40437–40441, 2003.