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

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 α 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 α 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 α 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 α 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 α 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. 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 α 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 α 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 α 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 α 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 α 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. 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 α subunit hydrolyzes ATP, becomes phosphorylated during the catalytic cycle, and transports the cations (1Kuhlbrandt W. Nat. Rev. Mol. Cell. Biol. 2004; 5: 282-295Crossref PubMed Scopus (434) Google Scholar). The β subunit is a molecular chaperone that is necessary for the correct membrane insertion of the α subunit and also modulates the transport properties of the α subunit (2Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (261) Google Scholar). Four α and 3 β isoforms have been identified that potentially can form 12 different isozymes with different transport and pharmacological properties (3Crambert G. Hasler U. Beggah A.T. Yu C. Modyanov N.N. Horisberger J.D. Lelievre L. Geering K. J. Biol. Chem. 2000; 275: 1976-1986Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 4Blanco G. Mercer R.W. Am. J. Physiol. 1998; 275: F633-F650PubMed Google Scholar). 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 α 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 (5Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). 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 (6Crambert G. Geering K. STKE's Science Genomics. 2003; (http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/166/re1)Google Scholar). 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.Sweadner, K. J., and Rael, E. (2000) 68, 41-56Google Scholar). FXYD2 or the γ subunit of Na,K-ATPase (8Forbush III, B. Kaplan J.H. Hoffman J.F. Biochemistry. 1978; 17: 3667-3676Crossref PubMed Scopus (221) Google Scholar) was the first FXYD protein that was identified as a specific modulator of renal Na,K-ATPase (9Béguin P. Wang X.Y. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (213) Google Scholar, 10Therien A.G. Goldshleger R. Karlish S.J. Blostein R. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar, 12Pu H.X. Cluzeaud F. Goldshlegger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Arystarkhova E. Wetzel R.K. Asinovski N.K. Sweadner K.J. J. Biol. Chem. 1999; 274: 33183-33185Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). It is now well established that also FXYD1 (phospholemman) (14Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (222) Google Scholar), a phospholemman-like protein from shark (15Mahmmoud Y.A. Cramb G. Maunsbach A.B. Cutler C.P. Meischke L. Cornelius F. J. Biol. Chem. 2003; 278: 37427-37438Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), FXYD4 (corticosteroid hormone-induced factor) (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar, 16Garty H. Lindzen M. Scanzano R. Aizman R. Fuzesi M. Goldshleger R. Farman N. Blostein R. Karlish S.J.D. Am. J. Physiol. 2002; 283: F607-F615Crossref PubMed Scopus (80) Google Scholar), and FXYD7 (17Béguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.-D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (117) Google Scholar) 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) 1The abbreviation used is: TM, transmembrane.1The abbreviation used is: TM, transmembrane. domains 8-10 (18Donnet C. Arystarkhova E. Sweadner K.J. J. Biol. Chem. 2001; 276: 7357-7365Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Moreover, a recent model (19Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A.B. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar), 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 (20Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar), predicts that FXYD2 is located in a pocket made up of TM9, TM6, TM4, and TM2 of the Na,K-ATPase α subunit. In this study, we investigated the role of TM9 of the Na,K-ATPase α subunit in the structural and functional interaction with FXYD proteins. For this purpose, we produced a model of the Na,K-ATPase α subunit to determine amino acids in the TM9 helix, which point to M2 and which could potentially interact with FXYD proteins. These amino acids or in by and the effects of these on the of association with different FXYD proteins and on functional effects in Xenopus that TM9 of the Na,K-ATPase α subunit is involved in the interaction with FXYD proteins. domains of interaction could identified that are 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 α subunit is by of the Na,K-ATPase α and Na,K-ATPase subunit was by to one at and point by the as by and PubMed Scopus Google Scholar). The of the TM9 was a using The of by for and subunits by J. The of the to of the Na,K-ATPase subunit and to of the α subunit has been K. Crambert G. Yu C. Modyanov N.N. Biochemistry. 2000; PubMed Scopus Google Scholar) in Xenopus and by in K. PubMed Scopus Google Scholar). Xenopus oocytes as K. Beggah A. P. S. S. D. P. J. Biol. PubMed Scopus (125) Google Scholar). with for or Na,K-ATPase subunits or for N.N. P. Beggah A.T. Horisberger J.D. Geering K. Am. J. Physiol. Google Scholar) or with for the Na,K-ATPase subunit or β subunit by G. with or for (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar), FXYD4 (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar), or FXYD7 (17Béguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.-D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (117) Google Scholar). in in the of protein at and to a and to and in each as K. Beggah A. P. S. S. D. P. J. Biol. PubMed Scopus (125) Google Scholar). and of between FXYD and and FXYD4 with or Na,K-ATPase subunits as K. Beggah A. P. S. S. D. P. J. Biol. PubMed Scopus (125) Google Scholar) by using an subunit M. Geering K. D. C. J. 20: Scopus Google Scholar). of FXYD7 by an α subunit is not very the association of FXYD7 with Na,K-ATPase was by using an FXYD7 (17Béguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.-D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (117) Google Scholar) The association of FXYD2 with and the was by using a K. Beggah A. P. S. S. D. P. J. Biol. PubMed Scopus (125) Google Scholar) proteins on and by by and as in the for the analysis was by functional effect of in the Na,K-ATPase subunit with or FXYD proteins was by the apparent affinity for K+ or Na+ of Na,K-ATPase. 3 of Xenopus oocytes with and or and or with FXYD by using the of the apparent K+ affinity as (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar) in the of which the but not the Na,K-ATPase. The and the apparent K+ affinity in the of by the to the using a of F. P. Geering K. Horisberger J.D. J. Physiol. PubMed Scopus Google Scholar). of the apparent Na+ affinity of Na,K-ATPase as U. Wang Crambert G. P. F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) in oocytes or Na,K-ATPase and subunits and the Na+ and γ subunits in the or of FXYD proteins. The was to the by using a of 3 U. Wang Crambert G. P. F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with a oocytes with an of and with an Na+ concentration of used for the of analysis was by on model of the J.D. S. Guennoun S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) in the a model of the was using an The of the and in In from an of the FXYD7 helix in the of TM2 and TM9 of the Na,K-ATPase α subunit, the FXYD7 using two or the FXYD7 helix a other to the or the of the of FXYD7. The and was a two high to a and of the FXYD7 each was a short energy of the FXYD7 helix as well as TM9 and TM2 was using the S. M. J. Chem. Scopus Google Scholar). The of of by of The of a was as its energy by using the D. M. J.D. S. J. H. S. D. L. K. C. S. B. B. M. R. J. M. J. D. M. J. Chem. B. 1998; PubMed Scopus Google Scholar) with a potential. was by of a in the model W. M. III, J. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, W. III, J. Chem. 2003; PubMed Scopus Google Scholar) with its used in the in the of the The during the based on and a was for each The of the Na,K-ATPase α in with have (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar) that not with FXYD2 or the of the Na,K-ATPase α subunit is involved in the interaction with FXYD2 as by (18Donnet C. Arystarkhova E. Sweadner K.J. J. Biol. Chem. 2001; 276: 7357-7365Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we produced a the sequence of the Na,K-ATPase subunit up to and that of the α subunit N.N. P. Beggah A.T. Horisberger J.D. Geering K. Am. J. Physiol. Google Scholar) TM9 and was in Xenopus oocytes with the β subunit and FXYD2, and the of FXYD2 association with this was with that with Na,K-ATPase subunit and with (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar), in oocytes the Na,K-ATPase and subunit and FXYD2, the β subunit and FXYD2 could with an against the Na,K-ATPase α subunit the other association of FXYD2 is in oocytes the a expression of FXYD2 The association of FXYD2 with the was by about and and that the of the Na,K-ATPase α subunit is involved in the stable association with with with the Na,K-ATPase subunit. Xenopus oocytes with Na,K-ATPase subunit, α subunit or with or β subunit in the or of FXYD2 for with and to and and in using a Na,K-ATPase subunit and or an on of in on are the of the α the and the β subunits and of of in A. are the between FXYD2 and the Na,K-ATPase subunit or the and of two with is The of TM9 of the Na,K-ATPase α in the with FXYD a model deduced from a crystallographic analysis of renal Na,K-ATPase predicts that TM9 of the α subunit is in to FXYD2 (19Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A.B. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar), we the role of TM9 in the structural and functional interaction with FXYD proteins. produced a model of the TM9 helix J.D. S. Guennoun S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and identified Phe956, Glu960, Leu964, and which could to FXYD proteins and potentially could form an interaction these amino acids of the located on the of the TM9 helix, or in with these α with subunits and FXYD2 in Xenopus and association with FXYD2 by with a Na,K-ATPase α subunit α to the α subunit, with the β subunit, and of the between FXYD2 and the α subunit show that the and the not the stable association of the other the and the the association a or a stable association of FXYD2 with these α The in which amino acids by the association of FXYD2 up to a to FXYD2, FXYD4 and with the but not with the FXYD7 C and association was efficient with the a with the α subunit and by and with the a and these that TM9 of the Na,K-ATPase α subunit and in Leu964 and/or Phe967 contribute to the stable interaction with FXYD of the Na,K-ATPase subunit contributes to the stable association with FXYD4 and FXYD7. and Xenopus oocytes with for or Na,K-ATPase subunits and subunit with FXYD4 or FXYD7 and for with and to and and using a Na,K-ATPase subunit and or an FXYD7 on are the of the α subunit, the and the β subunits and of FXYD4 and FXYD7. of in A. are the between FXYD4 and the α subunit. The between FXYD4 and subunit of was to of in C. is the of α subunit by an FXYD7 for the of α subunit as by with an α subunit The of α subunit by an FXYD7 a was to are of The of TM9 of the Na,K-ATPase α in the of FXYD in the the and the α an effect on the of the for K+ The and the the the as with the subunit, and the a effect at very membrane Glu960 in the has been to of the of Na,K-ATPase R. D. J. W. Karlish S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), by not importance of this amino in the of Na,K-ATPase Cell. Mol. Biol. Google Scholar). FXYD2 (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar), FXYD4 (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar), and FXYD7 (17Béguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.-D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (117) Google Scholar) have distinct effects on the of the apparent K+ affinity of Na,K-ATPase and investigated TM9 of the Na,K-ATPase α subunit is in the functional effect of FXYD proteins on the of the Na,K-ATPase. FXYD2, with the the or the a functional effect on the of the of Na,K-ATPase with the other FXYD2 was with the α its functional effect on the of the of Na,K-ATPase was with Phe956 and Glu960 in TM9 of the Na,K-ATPase α subunit are also important for the K+ effect of FXYD4 and FXYD7. The the functional effect of FXYD4 with and that of FXYD7 with also investigated the relative contribution of Phe956 and Glu960 to the K+ effect of the three FXYD proteins. Significantly, the to the of the functional effect of FXYD2 and FXYD4 and to a the and the other the a functional effect of FXYD7 whereas the or the functional effect of FXYD7 on the of the K+ affinity of Na,K-ATPase Phe956 and/or Glu960, which not contribute to the stable interaction of FXYD proteins with the Na,K-ATPase α subunit, are involved in the functional effect of FXYD proteins on the apparent K+ affinity of the Na,K-ATPase. FXYD proteins such as FXYD2 and FXYD4 have not an effect on the apparent affinity of Na,K-ATPase for K+ but also on the apparent affinity for intracellular Na+ (11Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.-D. Geering K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (131) Google Scholar). in FXYD2 whereas FXYD4 increases the apparent Na+ affinity of the Na,K-ATPase. investigated Phe956 and Glu960 in the Na,K-ATPase α subunit are involved not in the K+ effect but also in the Na+ effect of FXYD2 and FXYD2 or FXYD4 was with the α the of the Na,K-ATPase to a as FXYD2 or FXYD4 was with the Na,K-ATPase α subunit that Phe956 and Glu960 not contribute to the functional effect of FXYD proteins on the apparent Na+ affinity of Na,K-ATPase. of FXYD7 on of the α docking of FXYD7 on the model of the Na,K-ATPase α subunit J.D. S. Guennoun S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) was by a during the of from which the with the energy and based on the of the a energy of and the of the a energy of the of FXYD7 in the the with and of with Phe967 from TM9 FXYD7 interacts with Phe956, Glu960, Leu964, and Phe967 and from TM9 and and from of FXYD7 and are in the is to Leu964 and and for in each with interactions with Phe956 or to also interacts with of and the of the by with and from or of the from from the docking with Na,K-ATPase α in a and protein modeling, we provide for an interaction of FXYD proteins with TM9 of the which contributes to the efficient association and the functional effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. on crystallographic analysis of renal by (19Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A.B. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar) that FXYD2 or the γ subunit is located in a pocket made up of TM9, TM6, TM4, and TM2 of the Na,K-ATPase α subunit. on this and using the structure of the Ca-ATPase (20Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar), we produced a model of TM9 J.D. S. Guennoun S. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and amino acids that point to M2 and could potentially interact with FXYD proteins. we that of these amino acids by could have effects on the of the Na,K-ATPase α subunit, suggest that the of the TM9 helix of the Na,K-ATPase α subunit at one of the interaction with FXYD proteins. amino acids are well conserved in different Na,K-ATPase α which is with the that expression in Xenopus FXYD proteins with α isoforms (9Béguin P. Wang X.Y. Firsov D. Puoti A. Claeys D. Horisberger J.D. Geering K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (213) Google Scholar, P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.-D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (117) Google Scholar). Significantly, in the interaction face of TM9, we can with different functional The contributes to the stable interaction between the Na,K-ATPase α subunit and FXYD proteins but not to the functional effect of FXYD proteins on the apparent K+ affinity of Na,K-ATPase. The of a of the functional effect of FXYD proteins a of the association in the can one that the not the of protein in the cell but an the the interaction with FXYD proteins, is that not TM9 but also other of the TM9, TM6, TM4, and TM2 pocket in the Na,K-ATPase α subunit contribute to the structural interaction of FXYD proteins. is also by the that in and α amino acids at the of Leu964 and Phe967 in Na,K-ATPase are not distinct to FXYD proteins not with these In contrast to the the not contribute to the stable interaction between Na,K-ATPase and FXYD proteins, but the K+ effect of FXYD proteins to Na,K-ATPase. of by the effect of FXYD2 and FXYD7 on the apparent K+ affinity of Na,K-ATPase and that of hormone-induced Moreover, is to that the contribution of Phe956 and Glu960 to the K+ effect of different FXYD proteins is the of the effect of are complex, is that the contributes to the of the functional effect of FXYD2, but the functional effect of FXYD7. The of amino acids in the TM9 helix of the Na,K-ATPase α subunit in the K+ effect of FXYD proteins is to the intrinsic differences in the of the apparent K+ affinity of Na,K-ATPase by different FXYD proteins. FXYD proteins in and but show a sequence in the specific sequence differences in the domain of FXYD proteins that could interactions with TM9 of the Na,K-ATPase α subunit, in different functional In contrast to the K+ the Na+ effect of FXYD proteins such as FXYD2 and FXYD4 is not by Phe956 and Glu960 in the TM9 of the Na,K-ATPase α subunit. recent using proteins between FXYD2 and FXYD4 has that the transmembrane of these FXYD proteins determine the effect of FXYD2 and FXYD4 on the apparent Na+ affinity of the Na,K-ATPase M. Aizman R. Karlish S.J.D. Garty H. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). with these suggest that not the structural but also the functional interaction with FXYD proteins is by interaction The model of FXYD7 with the Na,K-ATPase α subunit in the is in agreement with the in TM9, Phe956, Glu960, Leu964, and identified by the analysis are involved in between FXYD7 and the Na,K-ATPase α subunit in the In the model predicts and in TM2 and and in FXYD7 as interaction the interactions Leu964 and Phe967 in TM9 are in and in the of which has been to the association with Na,K-ATPase G. C. Geering K. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), not to form by the It to of these could correct of FXYD proteins, to investigated a between the of in A.G. J. Mol. Biol. 2002; PubMed Scopus Google Scholar) and the association of in FXYD7 as well as in FXYD2 H.X. Scanzano R. Blostein R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the by the docking such and in the Na,K-ATPase α subunit, and the amino acids in are good for and for