JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M406697200 on July 2, 2004

J. Biol. Chem., Vol. 279, Issue 37, 38895-38902, September 10, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/37/38895    most recent
M406697200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, C.
Right arrow Articles by Geering, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, C.
Right arrow Articles by Geering, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins*

Ciming Li{ddagger}, Aurelien Grosdidier§, Gilles Crambert{ddagger}, Jean-Daniel Horisberger{ddagger}, Olivier Michielin§, and Käthi Geering{ddagger}

From the {ddagger}Department of Pharmacology and Toxicology of the University, Rue du Bugnon 27, CH-1005 Lausanne and the §Ludwig Institute for Cancer Research Lausanne Branch and Swiss Institute of Bioinformatics, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland

Received for publication, June 16, 2004 , and in revised form, June 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K+ and Na+ affinity. Little is known about the interaction sites between the Na,K-ATPase {alpha} subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase {alpha} subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe956, Glu960, Leu964, and Phe967 in TM9 of the Na,K-ATPase {alpha} subunit represent one face interacting with the three FXYD proteins. Leu964 and Phe967 contribute to the efficient association of FXYD proteins with the Na,K-ATPase {alpha} subunit, whereas Phe956 and Glu960 are essential for the transmission of the functional effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. The relative contribution of Phe956 and Glu960 to the K+ effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. In contrast to the effect on the apparent K+ affinity, Phe956 and Glu960 are not involved in the effect of FXYD2 and FXYD4 on the apparent Na+ affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe956, Glu960, Leu964, and Phe967 in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase {alpha} subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Na,K-ATPase transports three Na+ against two K+ across the plasma membrane of animal cells by using the energy of the hydrolysis of ATP. Its main task is the maintenance of the transmembrane Na+ and K+ gradients that permit the maintenance of the cell volume and the membrane potential. Moreover, Na+ gradients provide the energy for many secondary transport systems of vital importance. In addition to these basic functions, Na,K-ATPase is involved in many specialized tissue functions such as transepithelial Na+ transport and muscle and neuronal excitability.

Na,K-ATPase is an oligomeric protein. The {alpha} subunit hydrolyzes ATP, becomes phosphorylated during the catalytic cycle, and transports the cations (1). The {beta} subunit is a molecular chaperone that is necessary for the correct membrane insertion of the {alpha} subunit and also modulates the transport properties of the {alpha} subunit (2). Four {alpha} and 3 {beta} isoforms have been identified that potentially can form 12 different isozymes with different transport and pharmacological properties (3, 4).

In agreement with its important physiological role, Na,K-ATPase is finely regulated. Established regulatory mechanisms include changes in the intracellular Na+ concentration that produce short term regulation of the Na,K-ATPase transport rate, phosphorylation of the {alpha} subunit by cAMP-dependent protein kinase and protein kinase C that influences the distribution of Na,K-ATPase between the plasma membrane and intracellular stores, and long term regulation that increases the number of Na,K-ATPase units (5). Recently, a novel regulatory mechanism has been identified that involves tissue- and isozyme-specific interactions between Na,K-ATPase and small membrane proteins of the FXYD protein family (6).

The FXYD protein family contains seven members that are characterized by one transmembrane domain and a signature sequence that contains the FXYD motif and 3 other conserved amino acids (7). FXYD2 or the {gamma} subunit of Na,K-ATPase (8) was the first FXYD protein that was identified as a specific modulator of renal Na,K-ATPase (9-13). It is now well established that also FXYD1 (phospholemman) (14), a phospholemman-like protein from shark (15), FXYD4 (corticosteroid hormone-induced factor) (11, 16), and FXYD7 (17) also play a tissue-specific role in Na,K-ATPase regulation. Significantly, each of these auxiliary subunits produce a distinct functional effect on Na,K-ATPase that is adapted to the physiological needs of the tissues in which they are expressed.

The functional effects of FXYD proteins on Na,K-ATPase have been studied extensively, but the molecular basis of these effects is unknown, and very little is known on the interaction sites in the Na,K-ATPase and the FXYD proteins that mediate the efficient association between these two proteins and transmit the functional effects of FXYD proteins on Na,K-ATPase. Experiments based on thermal denaturation suggest that the association of FXYD2 occurs with transmembrane (TM)1 domains 8-10 (18). Moreover, a recent model (19), deduced from an electron crystallographic analysis at 9.5-Å resolution of renal Na,K-ATPase and by taking as a basis the high resolution structure of the Ca-ATPase (20), predicts that FXYD2 is located in a pocket made up of TM9, TM6, TM4, and TM2 of the Na,K-ATPase {alpha} subunit. In this study, we investigated the role of TM9 of the Na,K-ATPase {alpha} subunit in the structural and functional interaction with FXYD proteins. For this purpose, we produced a model of the Na,K-ATPase {alpha} subunit to determine amino acids in the TM9 helix, which point to M2 and which could potentially interact with FXYD proteins. These amino acids were substituted individually or in combination by alanines, and the effects of these mutations on the efficiency of association with different FXYD proteins and on their functional effects were tested after co-expression in Xenopus oocytes.

Our results indicate that TM9 of the Na,K-ATPase {alpha} subunit is involved in the interaction with FXYD proteins. Different domains of interaction could be identified that are either involved in the stable association with FXYD proteins or involved in the functional effect produced by FXYD proteins on the apparent K+ affinity of Na,K-ATPase. The interaction of FXYD proteins with TM9 of the Na,K-ATPase {alpha} subunit is supported by modeling of the Na,K-ATPase {alpha} subunit/FXYD7 complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis and Chimeras—The rat Na,K-ATPase {alpha}1 subunit was modified by silent mutagenesis to introduce one restriction site (AccI) at nucleotide position 2893, and then point mutations were introduced by the PCR-based method as described by Nelson and Long (21). The insert of the TM9 was subcloned into a pSD5 vector using AccI/DraIII restriction sites. The nucleotide sequences of all constructs were confirmed by dideoxy sequencing. cDNAs for rat {alpha}1 and {beta}1 subunits were kindly provided by J. Lingrel. The construction of the chimera {alpha}1-AL1 containing Met1 to Arg941 of the human Na,K-ATPase {alpha}1 subunit and Asn960 to Tyr1039 of the human non-gastric H,K-ATPase {alpha} subunit (AL1) has been described previously (22)

Protein Expression in Xenopus Oocytes and Metabolic Labeling—cRNAs were obtained by in vitro transcription (23). Stage V-VI Xenopus oocytes were obtained as described (24). Oocytes were injected with cRNAs coding for wild type or mutant rat Na,K-ATPase {alpha}1 subunits (10 ng/oocyte) or for AL1 (10 ng/oocyte) (25) or {alpha}1-AL1 (10 ng/oocyte) together with cRNAs for the rat Na,K-ATPase {beta}1 subunit (1 ng/oocyte) or rabbit gastric H,K-ATPase {beta} subunit (1 ng/oocyte) (kindly provided by G. Sachs) with or without cRNAs coding for human FXYD2a (11), rat FXYD4 (11), or mouse FXYD7 (2 ng) (17). Oocytes were incubated in modified Barth's solution in the presence of 0.8-1 mCi/ml [35S]methionine (Easy Tag Express [35S] protein labeling kit, PerkinElmer Life Sciences) at 19 °C and subjected to a 6-h pulse and to 24-, 48-, and 72-h chase periods in modified Barth's solution containing 10 mM cold methionine. Microsomes were prepared after each chase period as described previously (24).

Immunoprecipitation and Quantification of Association between FXYD Proteins and Na,K-ATPase—FXYD2 and FXYD4 were co-immunoprecipitated with wild type or mutant Na,K-ATPase {alpha}1 subunits under non-denaturing conditions as described (24) by using an {alpha}1 subunit antibody (26). Since co-immunoprecipitation of FXYD7 by an {alpha} subunit antibody is not very efficient, the association of FXYD7 with Na,K-ATPase was tested by using an FXYD7 antibody (17) under non-denaturing conditions. The association of FXYD2 with AL1 and the {alpha}1-AL1 chimera was tested by using a AL1 antibody (24) under non-denaturing conditions. Immunoprecipitated proteins were loaded on SDS-polyacrylamide gels (5-13%) and revealed by fluorography. Proteins were quantified by laser densitometer (KB Ultrascan 2202), and calculations were performed as described in the legends for the figures. Statistical analysis was performed by unpaired Student's t tests.

Electrophysiological Measurements—The functional effect of mutations in the Na,K-ATPase {alpha}1 subunit expressed with or without FXYD proteins was assessed by studying the apparent affinity for external K+ or internal Na+ of Na,K-ATPase. Electrophysiological measurements were performed 3 days after injection of Xenopus oocytes with rat, wild type {alpha}1 and {beta}1, or mutant {alpha}1 and {beta}1 cRNAs alone or together with FXYD cRNAs by using the two-electrode voltage clamp technique. Measurements of the apparent external K+ affinity were carried out as described previously (11) in the presence of 1 µM ouabain, which inhibits the endogenous oocyte Na,K-pump but not the expressed ouabain-resistant rat Na,K-ATPase. The maximal Na,K-pump current and the apparent K+ affinity (K1/2K+), measured in the presence of 100 mM external Na+, were obtained by fitting the Hill equation to the data using a Hill coefficient of 1.6 (27). Measurements of the apparent Na+ affinity of Na,K-ATPase were performed as described previously (28) in oocytes co-expressing rat, wild type, or mutant Na,K-ATPase {alpha}1 and {beta}1 subunits and the rat epithelial Na+ channel {alpha}, {beta}, and {gamma} subunits in the presence or absence of FXYD proteins. The Hill equation was fitted to the experimental data by using a Hill coefficient of 3 (28). Curves with a correlation coefficient lower than 0.90 were rejected. Only oocytes with an Imax of more than 100 nA and with an initial internal Na+ concentration of less than 10 mM were used for the determination of K1/2Na+ values. Statistical analysis was performed by unpaired Student's t test.

Protein Modeling—Based on our previous homology model of the Bufo Na-K-ATPase (29) in the E1 conformation, a model of the FXYD7/Na-K-ATPase complex was built using an evolutionary algorithm. The details of the calculations will be presented separately.2 In brief, starting from an arbitrary conformation of the FXYD7 helix in the vicinity of TM2 and TM9 of the Bufo Na,K-ATPase {alpha} subunit, the FXYD7 coordinates were refined using two operators translating or rotating the FXYD7 helix around a random axis. Two other operators were designed to rationalize the search, performing rotations around or translations along the axis of the modeled fragment of FXYD7. The fifth and last operator was a semistochastic interpolator combining two high scoring complexes to generate a new position and orientation of the FXYD7 fragment. After each operator was applied, a short energy minimization of the FXYD7 helix as well as TM9 and TM2 residues was performed using the CHARMM program (30). The minimization consisted of 30 steps of steepest descents followed by 50 steps of Adopted Basis Newton-Raphson.

The fitness of a complex was defined as its total enthalpic energy calculated by CHARMM using the CHARMM22 (31) parameters with a soft Van Der Waals potential. An implicit lipid layer was added by means of a dielectric switch in the Generalized Born-Simple Switch solvation model (32, 33) with its default parameter set. No mutation data were used in the conformational space search nor in the ranking of the complexes. The best scoring complexes generated during the evolutionary search were clustered, based on heavy atom root mean square difference values, and a contact list was generated for each cluster.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The TM9-TM10 Region of the Na,K-ATPase {alpha} Subunit Is Involved in Interaction with FXYD2—We have previously shown (11) that non-gastric H,K-ATPase does not associate with FXYD2 or FXYD4. To test whether the TM8-TM10 region of the Na,K-ATPase {alpha} subunit is involved in the interaction with FXYD2 as suggested by biochemical evidence (18), we produced a chimera containing the sequence of the Na,K-ATPase {alpha}1 subunit up to TM8 and that of the human non-gastric H,K-ATPase {alpha} subunit (AL1) (25) including TM9 and TM10 (Fig. 1). This chimera ({alpha}1-AL1) was expressed in Xenopus oocytes together with the H,K-ATPase {beta} subunit and FXYD2, and the efficiency of FXYD2 association with this chimera was compared with that with wild type Na,K-ATPase {alpha}1 subunit and with AL1. As shown previously (11), in metabolically labeled oocytes expressing the Na,K-ATPase {alpha}1 and {beta}1 subunit and FXYD2, the {beta} subunit and FXYD2 could be co-immunoprecipitated with an antibody against the Na,K-ATPase {alpha} subunit over prolonged chase periods (Fig. 2A, lanes 4-6). On the other hand, no association of FXYD2 is observed in oocytes co-expressing the wild type AL1 (lanes 7-9) despite a similar expression of FXYD2 (Fig. 2B, lanes 4-9). The association efficiency of FXYD2 with the chimeric {alpha}1-AL1 was reduced by about 70% (Fig. 2A, lanes 10-12, and 2C) after 48- and 72-h chase periods, indicating that the TM9-TM10 region of the Na,K-ATPase {alpha} subunit is involved in the stable association with FXYD2.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 1.
{alpha}-mutants and chimera. A, linear models of the Na,K-ATPase {alpha}1 subunit, the non-gastric H,K-ATPase {alpha} subunit (AL1), and the {alpha}1-AL1 chimera in which the TM domain 9 and 10 and C terminus of the human Na,K-ATPase {alpha}1 subunit were replaced by those of the human non-gastric H, K-ATPase. Indicated is Met1 and Arg941 of the Na,K-ATPase {alpha}1 subunit and Asn960 and Tyr1039 of AL1, which are comprised in the {alpha}1-AL1 chimera. Black and hatched rectangles indicate the TM domains of the Na,K-ATPase {alpha}1 subunit and non-gastric H,K-ATPase {alpha} subunit, respectively. B, amino acid sequences of TM9 of rat, wild type (WT), and mutant Na,K-ATPase {alpha}1 subunit.

 



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 2.
FXYD2 associates less efficiently with {alpha}1-AL1 than with the wild type Na,K-ATPase {alpha}1 subunit. A, Xenopus oocytes were injected with rat Na,K-ATPase {alpha}1 subunit, human non-gastric H,K-ATPase {alpha} subunit (AL1), or {alpha}1-AL1 chimera cRNAs (10 ng) together with rat {beta}1 cRNA (1 ng) or rabbit gastric H,K-ATPase {beta} subunit cRNA (1 ng) in the presence (lanes 4-12) or absence (lanes 1-3) of human FXYD2 cRNA (2 ng). Oocytes were metabolically labeled for 6 h with [35S]methionine and subjected to 24-, 48-, and 72-h chase periods. Microsomes were prepared, and immunoprecipitations were performed in non-denaturing conditions using a Na,K-ATPase {alpha}1 subunit antibody (lanes 1-6 and lanes 10-12) or an AL1 antibody (lanes 7-9). Immunoprecipitates were migrated on SDS-polyacrylamide gels. B, aliquots of samples described in A were loaded on SDS-polyacrylamide gels without immunoprecipitation. Indicated are the positions of the {alpha} subunits, the core-glycosylated (c{beta}), and the fully glycosylated (f{beta}) {beta} subunits and of FXYD2. C, quantification of data shown in A. Shown are the ratios between FXYD2 and the Na,K-ATPase {alpha}1 subunit (black bars) or the {alpha}1AL1 chimera (white bars) after 48- and 72-h chase periods. One out of two experiments with similar results is shown.

 
The Role of TM9 of the Na,K-ATPase {alpha} Subunit in the Stable Interaction with FXYD Proteins—Since a model deduced from a crystallographic analysis of renal Na,K-ATPase predicts that TM9 of the {alpha} subunit is in close proximity to FXYD2 (19), we tested the role of TM9 in the structural and functional interaction with FXYD proteins. We produced a model of the TM9 helix (29) and identified Ile953, Phe956, Glu960, Leu964, and Phe967, which could be directed to FXYD proteins and potentially could form an interaction face. We substituted these amino acids of the rat {alpha}1 isoform, located on the same side of the TM9 helix, either individually or in combination with alanine residues (Fig. 1), expressed these {alpha} mutants together with rat {beta}1 subunits and FXYD2 in Xenopus oocytes, and tested their association efficiency with FXYD2 by performing non-denaturing immunoprecipitations with a Na,K-ATPase {alpha} subunit antibody (Fig. 3A). All {alpha} mutants were synthesized similar to the wild type {alpha} subunit, associated with the {beta} subunit, and became stabilized over prolonged chase periods. Determinations of the ratios between associated FXYD2 and the {alpha} subunit show that the I/A mutant (Fig. 3Ba) and the FE/AA mutant (Fig. 3Bd) did not affect the stable association of FXYD2. On the other hand, the LF/AA (Fig. 3Bb) and the ILF/AA (Fig. 3Bc) significantly reduced the association efficiency after a 48- or 72-h chase period, indicating a less stable association of FXYD2 with these {alpha} mutants. The 5A (Fig. 3Be) mutant, in which all 5 amino acids were replaced by alanines, reduced the association efficiency of FXYD2 up to 50% after a 72-h chase period. Similar to FXYD2, FXYD4 (Fig. 4, A and B) associated efficiently with the FE/AA mutant but not with the ILF/AAA mutant. FXYD7 (Fig. 4, C and E) association was slightly less efficient with the FE/AA mutant after a 72-chase period than with the wild type {alpha} subunit and decreased by 44 and 54% with the ILF/AAA mutant after a 48- and 72-h chase period, respectively. Altogether, these results indicate that TM9 of the Na,K-ATPase {alpha} subunit and in particular Leu964 and/or Phe967 contribute to the stable interaction with FXYD proteins.



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 3.
TM9 of the Na,K-ATPase {alpha}1 subunit contributes to the stable association with FXYD2. A, Xenopus oocytes were injected with cRNAs for wild type (WT) or mutant rat Na,K-ATPase {alpha}1 subunits (10 ng) and rat {beta}1 subunit (1 ng) together with human FXYD2 (2 ng), metabolically labeled for 6 h with [35S]methionine, and subjected to 24-, 48-, and 72-h chase periods. Microsomes were prepared, and immunoprecipitations were performed under non-denaturing conditions using a Na,K-ATPase {alpha}1 subunit antibody. Immunoprecipitates were migrated on SDS-polyacrylamide gels. Indicated are the positions of the {alpha} subunit, the core-glycosylated (c {beta}), and the fully glycosylated (f {beta}) {beta} subunits and of FXYD2. B, quantification of data shown in A. Shown are the ratios between FXYD2 and the {alpha} subunit. The ratio between FXYD2 and wild type {alpha}1 subunit after 24 h of chase was arbitrarily set to 1. Closed squares, FXYD2/wild type {alpha}1; open squares, FXYD2/mutant {alpha}1. a, I/A mutant; b, LF/AA mutant; c, ILF/AAA mutant; d, FE/AA mutant; e, 5A mutant. Data are means ± S.E. of 4-8 experiments. *, = p < 0.05.

 



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 4.
TM9 of the Na,K-ATPase {alpha}1 subunit contributes to the stable association with FXYD4 and FXYD7. A, C, and D, Xenopus oocytes were injected with cRNAs for wild type (WT) or mutant rat Na,K-ATPase {alpha}1 subunits (10 ng) and rat {beta}1 subunit (1 ng) together with FXYD4 (2 ng) (A) or FXYD7 (2 ng) (C and D), metabolically labeled for 6 h with [35S]methionine, and subjected to 24-, 48-, and 72-h chase periods. Microsomes were prepared, and immunoprecipitations were performed under non-denaturing conditions using a Na,K-ATPase {alpha}1 subunit antibody (A and D) or an FXYD7 antibody (C). Immunoprecipitates were migrated on SDS-polyacrylamide gels. Indicated are the positions of the {alpha} subunit, the core-glycosylated (c{beta}), and the fully glycosylated (f{beta}) {beta} subunits and of FXYD4 and FXYD7. B, quantification of data shown in A. Shown are the ratios between FXYD4 and the {alpha} subunit. The ratio between FXYD4 and wild type {alpha}1 subunit after 24 h of chase was arbitrarily set to 1. Closed squares, FXYD4/wild type {alpha}1; open squares, FXYD4/mutant {alpha}1. a, FE/AA mutant; b, ILF/AAA mutant. E, quantification of data shown in C. Shown is the amount of {alpha} subunit co-immunoprecipitated by an FXYD7 antibody corrected for the total amount of {alpha} subunit expressed as revealed by immunoprecipitations with an {alpha} subunit antibody (D). The amount of {alpha} subunit co-immunoprecipitated by an FXYD7 antibody after a 24-h chase was arbitrarily set to 1. Closed squares, wild type {alpha}1; open squares, mutant {alpha}1. a, FE/AA mutant; b, ILF/AAA mutant. Data are means ± S.E. of 3-4 experiments. *, = p < 0.05.

 
The Role of TM9 of the Na,K-ATPase {alpha} Subunit in the Functional Effect of FXYD Proteins—As shown in Fig. 5, the I/A, the IL/AA, and the ILF/AAA {alpha} mutants had an effect per se on the voltage dependence of the K1/2 value for K+ (Fig. 5A). The FE/AA and the E/A mutants slightly increased the K1/2K+ over the whole potential range tested as compared with the wild type {alpha}1 subunit, and the F/A mutant showed a pronounced effect at very negative membrane potentials (Fig. 5B). Interestingly, Glu960 in the FE/AA mutant has been suggested to be part of the cation "occlusion" gate of Na,K-ATPase (34), although substitution by alanine did not reveal any importance of this amino acid in the cation dependence of Na,K-ATPase activity (35).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 5.
Effects of alanine substitutions in TM9 of the {alpha}1 subunit on functional properties of Na,K-ATPase. A and B, oocytes were injected with cRNAs for rat, wild type (WT), or mutant Na,K-ATPase {alpha}1 subunits (10 ng) and for rat {beta}1 subunit (1 ng). Three days after cRNA injection, K1/2K+ values of the Na,K-ATPase as a function of the membrane potential were determined as described under "Materials and Methods." Inset, maximal Na,K-pump currents (Imax). For clarity, no symbols for significance are indicated. In A, p < 0.01, wild type (closed squares) versus I/A (open squares), IL/AA (open triangles) and ILF/AAA (open circles) mutants between -130 mV and -50 mV, versus I/A and ILF/AAA mutants between 10 mV and 30 mV. In B, p < 0.01, wild type (closed squares) versus FE/AA (open squares) at all membrane potentials except at -130 mV and 30 mV; p < 0.01, wild type versus F/A (open triangles) at all membrane potentials; p < 0.05, wild type versus E/A (open circles) at all membrane potentials except at -130 mV.

 
FXYD2 (11), FXYD4 (11), and FXYD7 (17) have distinct effects on the voltage dependence of the apparent K+ affinity of Na,K-ATPase (Fig. 7Aa, 7Ba, and 7Ca). We investigated whether TM9 of the Na,K-ATPase {alpha} subunit is implicated in the functional effect of FXYD proteins on the K1/2K+ value of the Na,K-ATPase.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7.
Phe956 and/or Glu960 in TM9 of the Na,K-ATPase {alpha}1 subunit are involved in the functional effect of FXYD2, FXYD4, and FXYD7 on the apparent K+ affinity of Na,K-ATPase. Oocytes were injected with cRNAs for wild type (WT) or mutant rat Na,K-ATPase {alpha}1 subunit (10 ng) and the rat {beta}1 subunit (1 ng) together with cRNAs for FXYD2 (A), FXYD4 (B), or FXYD7 (C). Three days after cRNA injection, K1/2K+ values of the Na,K-ATPase as a function of the membrane potential were determined. Closed squares, wild type {alpha}1 subunit (Aa, Ba, Ca), FE/AA (Ab, Bb, Cb), F/A (Ac, Bc, Cc), or E/A (Ad, Bd, Cd) mutants expressed without FXYD protein; open squares, wild type or mutant {alpha}1 subunits expressed with FXYD proteins, with FXYD2 (A), with FXYD4 (B), or with FXYD7 (C). Imax values were 323.5 ± 22.4 nA, 455.6 ± 23 nA, 252.3 ± 17.5 nA, and 262.7 ± 27 nA in oocytes injected with {alpha}/{beta}, {alpha}/{beta}/FXYD2, {alpha}/{beta}/FXYD4, and {alpha}/{beta}/FXYD7 cRNAs, respectively. Data are means ± S.E. of 9-40 oocytes from 2-6 different batches. *, = p < 0.05.

 
FXYD2, co-expressed with the I/A (Fig. 6b), the IL/AA (Fig. 6c), or the ILF/AAA (Fig. 6d) mutant, retained a functional effect on the voltage dependence of the K1/2K+ of Na,K-ATPase (compare Fig. 6, b-d, with 6a). On the other hand, when FXYD2 was co-expressed with the FE/AA {alpha} mutant, its functional effect on the voltage dependence of the K1/2K+ of Na,K-ATPase was completely lost (compare Fig. 7Ab with 7Aa). Phe956 and Glu960 in TM9 of the Na,K-ATPase {alpha} subunit are also important for the K+ effect of FXYD4 and FXYD7. The FE/AA mutant significantly decreased the functional effect of FXYD4 (compare Fig. 7Bb with 7Ba) and completely abolished that of FXYD7 (compare Fig. 7Cb with 7Ca). We also investigated the relative contribution of Phe956 and Glu960 to the K+ effect of the three FXYD proteins. Significantly, the E/A mutant contributed significantly to the abolishment of the functional effect of FXYD2 (Fig. 7Ad) and FXYD4 (Fig. 7Bd) and to a lesser extent, the F/A mutant (Fig. 7Ac and 7Bc). On the other hand, the E/A mutant permitted a full functional effect of FXYD7 (Fig. 7Cd), whereas the F/A mutant reduced or modified the functional effect of FXYD7 on the voltage dependence of the K+ affinity of Na,K-ATPase (Fig. 7Cc). Thus, Phe956 and/or Glu960, which do not contribute to the stable interaction of FXYD proteins with the Na,K-ATPase {alpha} subunit, are involved in the functional effect of FXYD proteins on the apparent K+ affinity of the Na,K-ATPase.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 6.
Ile953, Leu964,and Phe967 in TM9 of the Na,K-ATPase {alpha}1 subunit are not involved in the functional effect of FXYD2 on the apparent K+ affinity of Na,K-ATPase. Oocytes were injected with cRNAs for rat, wild type (wt), or mutant Na,K-ATPase {alpha}1 subunits (10 ng) and for rat {beta}1 subunit (1 ng) in the presence of cRNA for FXYD (2 ng). Three days after cRNA injection, K1/2K+ values of the Na,K-ATPase as a function of the membrane potential were determined. Closed squares, wild type {alpha}1 subunit (a), I/A (b), IL/AA (c), or ILF/AAA (d) mutants expressed without FXYD2; open squares, wild type or mutant {alpha}1 subunits expressed with FXYD2. Data are means ± S.E. of 11-40 oocytes from 3-6 different batches. *, = p < 0.05.

 
Some FXYD proteins such as FXYD2 and FXYD4 have not only an effect on the apparent affinity of Na,K-ATPase for extracellular K+ but also on the apparent affinity for intracellular Na+ (11). As shown in Fig. 8, FXYD2 decreases, whereas FXYD4 increases the apparent Na+ affinity of the Na,K-ATPase. We investigated whether Phe956 and Glu960 in the Na,K-ATPase {alpha} subunit are involved not only in the K+ effect but also in the Na+ effect of FXYD2 and FXYD4. When FXYD2 or FXYD4 was co-expressed with the FE/AA {alpha} mutant, the K1/2Na+ of the Na,K-ATPase decreased to a similar extent as when FXYD2 or FXYD4 was co-expressed with the wild type Na,K-ATPase {alpha} subunit (Fig. 8), indicating that Phe956 and Glu960 do not contribute to the functional effect of FXYD proteins on the apparent Na+ affinity of Na,K-ATPase.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 8.
Phe956 and/or Glu960 in TM9 of the Na,K-ATPase {alpha}1 subunit are not involved in the functional effect of FXYD2 and FXYD4 on the apparent Na+ affinity of Na,K-ATPase. Oocytes were injected with cRNAs for wild type (WT) or mutant rat Na,K-ATPase {alpha}1 subunits (10 ng) and the rat {beta}1 subunit (1 ng) alone or together with FXYD2 or FXYD4 (2 ng). After 2 days of incubation, oocytes were injected with rat epithelial Na+ channel cRNAs (0.5 ng of {alpha}, {beta}, and {gamma} subunit cRNAs). The measurement of the apparent affinity for internal Na+ of Na,K-ATPase was performed 1 day after injection of epithelial Na+ channel cRNA. Intracellular Na+-activated Na,K-pump currents were recorded with 100 mM extracellular Na+ at -50mV. *, = p < 0.05; **, = p < 0.001. Data are means ± S.E. of 9-27 oocytes from 2-7 different batches. CHIF, corticosteroid hormone-induced factor.

 
Docking of FXYD7 on Homology Model of the Na,k-ATPase {alpha} Subunit—The docking of FXYD7 on the previously described homology model of the Na,K-ATPase {alpha} subunit (29) was performed by a genetic algorithm. No mutation data were taken into account during the conformational space search. A total of 540 complexes were generated, from which the 10% complexes with the lowest energy were extracted and clustered based on heavy atom root mean square difference values (Fig. 9). Two clusters were identified; the cluster A (83% of the conformers) had a mean energy of -22676 kcal/mol, and the cluster B (17% of the conformers) had a mean energy of -22613 kcal/mol. Both clusters share the same anchoring of FXYD7 in the cleft near the extracellular space, with contacts involving residues Gln26 (cluster A) and Thr27 (cluster B) of FXYD7, with Phe967 from TM9 (Table I). FXYD7 interacts with residues Ile953, Phe956, Glu960, Leu964, and Phe967 and Leu968 from TM9 and Tyr149, Ile143, Arg156, and Ile157 from TM2. Two residues of FXYD7 (Met30 and Phe37) are overrepresented in the contact list. Met30 is close to both Leu964 and Leu968 (TM9) and Ile142 (TM2). Two alternative rotamers for Phe37 were isolated in each cluster with favorable interactions with either Phe956 (TM9) or Tyr149 (TM2). Next to Phe37, Val38 also interacts with Tyr149 (TM2). Two hydrophobic residues of FXYD7, Ile44 and Leu45, fill the widest part of the TM9-TM2 groove, stabilized by several contacts with Ile953 and Ile157 (structures from cluster B) or Arg156 (aliphatic part of the side chain, structures from cluster A).



View larger version (124K):
[in this window]
[in a new window]
  		FIG. 9.
Docking results. Left, overview of the Na,K-ATPase {alpha} subunit, showing TM2 and TM9, and two average structures from cluster A (green) and B (red). The location of the membrane is shown as transparent. Right, detailed view of the interaction between TM9 (left) and TM2 (right) and two average structures of FXYD7, from cluster A (green) and B (red). Heavy atoms of residues involved in the interaction are shown in blue for the Na,K-ATPase {alpha} subunit and in green or in red for cluster A and B.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Contact list derived from the docking calculations

See "Results" for details.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
By site-directed mutagenesis and protein modeling, we provide evidence for an interaction of FXYD proteins with TM9 of the Na,K-ATPase, which contributes to the efficient association and the functional effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. Based on crystallographic analysis of renal Na,K-ATPase, previous studies by Hebert et al. (19) predict that FXYD2 or the {gamma} subunit is located in a pocket made up of TM9, TM6, TM4, and TM2 of the Na,K-ATPase {alpha} subunit. Based on this hypothesis and using the structure of the sarco-endoplasmic reticulum Ca-ATPase pump (20), we produced a model of TM9 (29) and determined amino acids that point to M2 and could potentially interact with FXYD proteins. Although we cannot entirely exclude that substitution of these amino acids by alanine could have some indirect effects on the global conformation of the Na,K-ATPase {alpha} subunit, our results rather suggest that the predicted side of the TM9 helix of the Na,K-ATPase {alpha} subunit indeed represents at least one of the interaction faces with FXYD proteins. All amino acids tested are well conserved in different Na,K-ATPase {alpha} isoforms, which is compatible with the observation that after expression in Xenopus oocytes, FXYD proteins associate with all {alpha} isoforms (9, 17).

Significantly, in the interaction face of TM9, we can distinguish regions with different functional roles. The Leu964/Phe967 region contributes to the stable interaction between the Na,K-ATPase {alpha} subunit and FXYD proteins but not to the functional effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. The lack of a partial loss of the functional effect of FXYD proteins despite a partial loss of the association efficiency in the LF/AA mutant can be explained if one assumes that the co-immunoprecipitation experiments do not reflect the real amount of Na,K-ATPase/FXYD protein complexes in the cell but rather an increased detergent sensitivity. Since the LF/AA mutant only partially abolishes the interaction with FXYD proteins, it is likely that not only TM9 but also other TM helices of the TM9, TM6, TM4, and TM2 binding pocket in the Na,K-ATPase {alpha} subunit contribute to the structural interaction of FXYD proteins. This hypothesis is also supported by the fact that in non-gastric H,K-ATPase and gastric H,K-ATPase {alpha} subunits, amino acids at the positions of Leu964 and Phe967 in Na,K-ATPase are not distinct enough to explain why FXYD proteins do not associate with these P-type ATPases.

In contrast to the Leu964/Phe967 region, the Phe956/Glu960 region does not contribute to the stable interaction between Na,K-ATPase and FXYD proteins, but it transmits the K+ effect of FXYD proteins to Na,K-ATPase. Substitution of Phe956/Glu960 by alanines completely abolishes the effect of FXYD2 and FXYD7 on the apparent K+ affinity of Na,K-ATPase and strongly reduces that of corticosteroid hormone-induced factor. Moreover, it is interesting to note that the contribution of Phe956 and Glu960 to the K+ effect of different FXYD proteins is different. Although the precise interpretations of the effect of single mutations are complex, it is clear that the E/A mutant contributes significantly to the loss of the functional effect of FXYD2, but it permits the full functional effect of FXYD7. The differential roles of amino acids in the TM9 helix of the Na,K-ATPase {alpha} subunit in the K+ effect of FXYD proteins is likely to reflect the intrinsic differences in the modulation of the apparent K+ affinity of Na,K-ATPase by different FXYD proteins. FXYD proteins significantly differ in their N- and C-terminal region but show a significant sequence homology in the TM region. Nevertheless, specific sequence differences exist in the TM domain of FXYD proteins that could allow interactions with TM9 of the Na,K-ATPase {alpha} subunit, resulting in different functional effects.

In contrast to the K+ effect, the Na+ effect of certain FXYD proteins such as FXYD2 and FXYD4 is not mediated by Phe956 and Glu960 in the TM9 of the Na,K-ATPase {alpha} subunit. A recent study using chimeric proteins between FXYD2 and FXYD4 has revealed that the transmembrane segments of these FXYD proteins determine the opposite effect of FXYD2 and FXYD4 on the apparent Na+ affinity of the Na,K-ATPase (36). Together with our observations, these results suggest that not only the structural but also the functional interaction with FXYD proteins is determined by multiple interaction sites.

The model of FXYD7 docked with the Na,K-ATPase {alpha} subunit in the E1 conformation is in excellent agreement with the experimental data. All residues in TM9, Ile953, Phe956, Glu960, Leu964, and Phe967, identified by the mutagenesis analysis are involved in contacts between FXYD7 and the Na,K-ATPase {alpha} subunit in the model. In addition, the model predicts Ile142, Tyr149, Arg156, and Ile157 in TM2 and Gln26, Met30, Val38, Phe37, Ile41, Ile44, and Leu45 in FXYD7 as interaction sites. Interestingly, the stabilizing interactions involving Leu964 and Phe967 in TM9 are similar in both clusters. Gly29 and Gly40 in FXYD7, the substitution of which has previously been shown to significantly affect the association efficiency with Na,K-ATPase (37), were not predicted to form favorable contacts by the docking. It remains to be shown whether substitution of these glycine residues could perturb correct folding of FXYD proteins, thus limiting complex formation. Alternatively, it remains to be investigated whether there might be a correlation between the potential implication of Gly40 in oligomer formation (38) and the association efficiency of Gly40 mutants in FXYD7 as well as in FXYD2 (39). Finally, the residues suggested by the docking study only, such as, Tyr149, Ile157, Arg156, Ile142, and Leu968 in the Na,K-ATPase {alpha} subunit, and the predicted amino acids in FXYD7, are good candidates for further mutagenesis analysis.


   FOOTNOTES
 
* This study was supported by Grant 31-64793.01 from the Swiss National Science Foundation (to K. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 41-21-692-54-10; Fax: 41-21-692-53-55; E-mail: kaethi.geering{at}ipharm.unil.ch.

1 The abbreviation used is: TM, transmembrane. Back

2 A. Grosdidier and O. Michielin, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Sophie-Roy and Danièle Schaer for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kuhlbrandt, W. (2004) Nat. Rev. Mol. Cell. Biol. 5, 282-295[CrossRef][Medline] [Order article via Infotrieve]
  2. Geering, K. (2001) J. Bioenerg. Biomembr. 33, 425-438[CrossRef][Medline] [Order article via Infotrieve]
  3. Crambert, G., Hasler, U., Beggah, A. T., Yu, C., Modyanov, N. N., Horisberger, J. D., Lelievre, L., and Geering, K. (2000) J. Biol. Chem. 275, 1976-1986[Abstract/Free Full Text]
  4. Blanco, G., and Mercer, R. W. (1998) Am. J. Physiol. 275, F633-F650[Medline] [Order article via Infotrieve]
  5. Therien, A. G., and Blostein, R. (2000) Am. J. Physiol. 279, C541-C566
  6. Crambert, G., and Geering, K. (2003) STKE's Science Genomics http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/166/re1
  7. Sweadner, K. J., and Rael, E. (2000) 68, 41-56
  8. Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676[CrossRef][Medline] [Order article via Infotrieve]
  9. Béguin, P., Wang, X. Y., Firsov, D., Puoti, A., Claeys, D., Horisberger, J. D., and Geering, K. (1997) EMBO J. 16, 4250-4260[CrossRef][Medline] [Order article via Infotrieve]
  10. Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634[Abstract/Free Full Text]
  11. Béguin, P., Crambert, G., Guennoun, S., Garty, H., Horisberger, J.-D., and Geering, K. (2001) EMBO J. 20, 3993-4002[CrossRef][Medline] [Order article via Infotrieve]
  12. Pu, H. X., Cluzeaud, F., Goldshlegger, R., Karlish, S. J. D., Farman, N., and Blostein, R. (2001) J. Biol. Chem. 276, 20370-20378[Abstract/Free Full Text]
  13. Arystarkhova, E., Wetzel, R. K., Asinovski, N. K., and Sweadner, K. J. (1999) J. Biol. Chem. 274, 33183-33185[Abstract/Free Full Text]
  14. Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481[Abstract/Free Full Text]
  15. Mahmmoud, Y. A., Cramb, G., Maunsbach, A. B., Cutler, C. P., Meischke, L., and Cornelius, F. (2003) J. Biol. Chem. 278, 37427-37438[Abstract/Free Full Text]
  16. Garty, H., Lindzen, M., Scanzano, R., Aizman, R., Fuzesi, M., Goldshleger, R., Farman, N., Blostein, R., and Karlish, S. J. D. (2002) Am. J. Physiol. 283, F607-F615
  17. Béguin, P., Crambert, G., Monnet-Tschudi, F., Uldry, M., Horisberger, J.-D., Garty, H., and Geering, K. (2002) EMBO J. 21, 3264-3273[CrossRef][Medline] [Order article via Infotrieve]
  18. Donnet, C., Arystarkhova, E., and Sweadner, K. J. (2001) J. Biol. Chem. 276, 7357-7365[Abstract/Free Full Text]
  19. Hebert, H., Purhonen, P., Vorum, H., Thomsen, K., and Maunsbach, A. B. (2001) J. Mol. Biol. 314, 479-494[CrossRef][Medline] [Order article via Infotrieve]
  20. Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655[CrossRef][Medline] [Order article via Infotrieve]
  21. Nelson, R. M., and Long, G. L. (1989) Anal. Biochem. 180, 147-151[CrossRef][Medline] [Order article via Infotrieve]
  22. Geering, K., Crambert, G., Yu, C., Korneenko, T. V., Pestov, N. B., and Modyanov, N. N. (2000) Biochemistry 39, 12688-12698[CrossRef][Medline] [Order article via Infotrieve]
  23. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Nucleic Acids Res. 12, 7035-7056[Abstract/Free Full Text]
  24. Geering, K., Beggah, A., Good, P., Girardet, S., Roy, S., Schaer, D., and Jaunin, P. (1996) J. Cell Biol. 133, 1193-1204[Abstract/Free Full Text]
  25. Modyanov, N. N., Mathews, P. M., Grishin, A. V., Beguin, P., Beggah, A. T., Rossier, B. C., Horisberger, J. D., and Geering, K. (1995) Am. J. Physiol. 38, C992-C997
  26. Girardet, M., Geering, K., Frantes, J. M., Geser, D., Rossier, B. C., Kraehenbuhl, J.-P., and Bron, C. (1981) Biochem. J. 20, 6684-6691[CrossRef]
  27. Jaisser, F., Jaunin, P., Geering, K., Rossier, B. C., and Horisberger, J. D. (1994) J. Gen. Physiol. 103, 605-623[Abstract/Free Full Text]
  28. Hasler, U., Wang, X., Crambert, G., Beguin, P., Jaisser, F., Horisberger, J. D., and Geering, K. (1998) J. Biol. Chem. 273, 30826-30835[Abstract/Free Full Text]
  29. Horisberger, J. D., Kharoubi-Hess, S., Guennoun, S., and Michielin, O. (2004) J. Biol. Chem. 279, 29542-29550[Abstract/Free Full Text]
  30. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983) J. Comput. Chem. 4, 187-217[CrossRef]
  31. MacKerell, J., A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Jr., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, I., W.E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., and Karplus, M. (1998) J. Phys. Chem. B. 102, 3586-3616
  32. Im, W., Feig, M., and Brooks, C. L., III (2003) Biophys. J. 85, 2900-2918[Abstract/Free Full Text]
  33. Im, W., Lee, M. S., and Brooks, C. L., III (2003) J. Comput. Chem. 24, 1691-1702[CrossRef][Medline] [Order article via Infotrieve]
  34. Goldshleger, R., Tal, D., Moorman, J., Stein, W., and Karlish, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6911-6915[Abstract/Free Full Text]
  35. Van Huysse, J. W., and Lingrel, J. B. (1993) Cell. Mol. Biol. Res. 39, 497-507[Medline] [Order article via Infotrieve]
  36. Lindzen, M., Aizman, R., Lifshitz, Y., Lubarski, I., Karlish, S. J. D., and Garty, H. (2003) J. Biol. Chem. 278, 18738-18743[Abstract/Free Full Text]
  37. Crambert, G., Li, C., Swee, L. K., and Geering, K. (2004) J. Biol. Chem. 279, 30888-30895[Abstract/Free Full Text]
  38. Therien, A. G., and Deber, C. M. (2002) J. Mol. Biol. 322, 583-590[CrossRef][Medline] [Order article via Infotrieve]
  39. Pu, H. X., Scanzano, R., and Blostein, R. (2002) J. Biol. Chem. 277, 20270-20276[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Bibert, S. Roy, D. Schaer, J.-D. Horisberger, and K. Geering
Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes
J. Biol. Chem., January 4, 2008; 283(1): 476 - 486.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
I. Lubarski, S. J. D. Karlish, and H. Garty
Structural and functional interactions between FXYD5 and the Na+-K+-ATPase
Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1818 - F1826.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
D. Pavlovic, W. Fuller, and M. J. Shattock
The intracellular region of FXYD1 is sufficient to regulate cardiac Na/K ATPase
FASEB J, May 1, 2007; 21(7): 1539 - 1546.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
J. Y. CHEUNG, L. I. ROTHBLUM, J. R. MOORMAN, A. L. TUCKER, J. SONG, B. A. AHLERS, L. L. CARL, J. WANG, and X.-Q. ZHANG
Regulation of Cardiac Na+/Ca2+ Exchanger by Phospholemman
Ann. N.Y. Acad. Sci., March 1, 2007; 1099(1): 119 - 134.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Wang, X.-Q. Zhang, B. A. Ahlers, L. L. Carl, J. Song, L. I. Rothblum, R. C. Stahl, D. J. Carey, and J. Y. Cheung
Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na+/Ca2+ Exchanger
J. Biol. Chem., October 20, 2006; 281(42): 32004 - 32014.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Lifshitz, M. Lindzen, H. Garty, and S. J. D. Karlish
Functional Intera