Proc Natl Acad Sci U S A 99(17): 11476-11481

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

^{Institute of Pharmacology and Toxicology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland; and}^{Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel}

^{To whom reprint requests should be addressed. E-mail:}hc.linu.mrahpi@gnireeg.ihteak.

Edited by David H. MacLennan, University of Toronto, Toronto, ON, Canada, and approved July 8, 2002

Received 2002 May 3

Abstract

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different α-β isozymes of NKA after coexpression in Xenopus oocytes, and with α1–β, and less efficiently with α2–β isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K^{affinity of α1–β1 and α2–β1 isozymes and a nearly 2-fold decrease in the internal Na}^{affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.}

Abstract

The Na,K-ATPase (NKA) is an ubiquitous plasma membrane enzyme that transports 3 Na^{out and 2 K}^{into the cells by using the energy of the hydrolysis of ATP. NKA activity permits the creation and the maintenance of Na}^{and K}^{gradients across cell membranes that are essential for both cellular and body ion homeostasis. In addition to this functional role, NKA plays a role as a receptor for cardiac glycosides widely used in the treatment of heart failure because of their positive ionotropic action (}1) and presumably for endogenous digitalis-like compounds identified in mammals (2). The minimal functional unit of NKA comprises a catalytic α subunit containing the cation, ATP and phosphate binding sites, and a glycosylated β subunit required for the correct folding and functional maturation of the α subunit (3). Four α and 3 β isoforms, which may form different, tissue-specific NKA isozymes with distinct transport and pharmacological properties, have been identified (4, 5).

Regulation of NKA activity is an important and complex process that involves short- and long-term mechanisms. Short-term regulation of NKA is either mediated by changes in intracellular Na^{concentrations that directly affect the Na,K-pump activity or by peptide hormone-mediated phosphorylation/dephosphorylation reactions leading to changes in the Na,K-pump transport properties or in its cell surface expression. On the other hand, long-term regulation involves mineralocorticoid or thyroid hormone-mediated changes in the transcription of α- and/or β-subunit genes leading to an increased expression level of Na,K-pumps (}6). Recently, a new type of regulation has emerged that involves the association of small, single-span membrane proteins with NKA. These proteins belong to the so-called FXYD family, the members of which share a common signature sequences encompassing the transmembrane domain and adjacent regions (7). Three of the seven members of the FXYD family have so far been identified as regulators of NKA. Two FXYD2 variants (the so-called γa and γb subunits of NKA) (7–10) and FXYD4 (CHIF) (11, 12) were shown to be renal, nephron segment-specific regulators (13–16), whereas FXYD7 is a brain- and isozyme-specific regulator of NKA (44). The functional characteristics of three other members of this family, FXYD3 (MAT-8) (17), FXYD5 (RIC) (18), and FXYD6 (phosphohippolin) (19), have not yet been studied. Finally, the last member of the FXYD family, FXYD1, originally named phospholemman (PLM) (20), has been extensively investigated, but its actual physiological role remains obscure. FXYD1 is widely distributed with highest expression in heart and skeletal muscle (21), where it is the main substrate for protein kinase A and C (20). Expression of FXYD1 in oocytes or addition of the purified protein to lipid bilayers produces a chloride-sensitive current slowly activated by hyperpolarization (22). Moreover, PLM is able to switch among different conformations having different selectivities for cations and anions that permits the translocation of zwitterionic molecules such as taurine (23). More recently, Mahmmoud et al. (24) reported that a PLM-like protein coimmunoprecipitated with NKA from shark rectal glands. In view of these results, we tested whether, similar to FXYD2, -4, and -7, PLM is also a tissue-specific regulator of NKA. We demonstrate that PLM is indeed able to associate with NKA after coexpression in Xenopus oocytes as well as in native cardiac and skeletal muscle. PLM induces a small decrease in the apparent K^{affinity, as well as a 2-fold decrease in the apparent Na}^{affinity of NKA isozymes. The reduction in the apparent Na}^{affinity of NKA may be of physiological relevance during muscle contraction.}

Acknowledgments

We thank Danièle Schaer and Sophie Roy for excellent technical assistance. We also thank M. J. Caplan for the α1-antibody (6H), T. Pressley for the α1 and α2 isoform-specific antibodies, B. Attali for dog PLM cDNA, J. Lingrel for rat NKA α1, α2*, and α3* and β1 cDNAs, D. H. MacLennan for SERCA2a cDNAs, and D. Khananshvili for the bovine heart sarcolemma vesicles. This work was supported by grants from the Swiss National Fund for Scientific Research (31-64793.01 to K.G.) and the Roche Research Foundation (to K.G. and G.C.), and by a grant from the Minerva Foundation (to S.K. and H.G.).

Acknowledgments

Abbreviations

NKA, Na,K-ATPase
PLM, phospholemman
ER, endoplasmic reticulum
BFA, brefeldin A

Abbreviations

Notes

This paper was submitted directly (Track II) to the PNAS office.

Notes

This paper was submitted directly (Track II) to the PNAS office.

References

1. Gheorghiade M. & Pitt, B. (1997) Am. Heart J.134, 3-12. [[PubMed]
2. Hamlyn J. M., Hamilton, B. P. & Manunta, P. (1996) J. Hypertens.14, 151-167. [[PubMed]
3. Geering K(2001) J. Bioenerg. Biomembr.33, 425-438. [[PubMed][Google Scholar]
4. Crambert G., Hasler, U., Beggah, A. T., Yu, C., Modyanov, N. N., Horisberger, J. D., Lelièvre, L. & Geering, K. (2000) J. Biol. Chem.275, 1976-1986. [[PubMed]
5. Blanco G. & Mercer, R. W. (1998) Am. J. Physiol.275, F633-F650. [[PubMed]
6. Feraille E. & Doucet, A. (2001) Physiol. Rev.81, 345-418. [[PubMed]
7. Sweadner K. J. & Rael, E. (2000) Genomics68, 41-56. [[PubMed]
8. Forbush B., Kaplan, J. H. & Hoffman, J. F. (1978) Biochemistry17, 3667-3676. [[PubMed]
9. Mercer R. W., Biemesderfer, D., Bliss, D. P., Collins, J. H. & Forbush, B. (1993) J. Cell Biol.121, 579-586.
10. Kuster B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M. & Karlish, S. J. (2000) J. Biol. Chem.275, 18441-18446. [[PubMed]
11. Attali B., Latter, H., Rachamim, N. & Garty, H. (1995) Proc. Natl. Acad. Sci. USA92, 6092-6096.
12. Garty, H., Lindzen, M., Scanfano, R., Aizman, R., Füzesi, M., Goldshleger, R., Farman, N., Blostein, R. & Karlish, S. J. D. (2002) Am. J. Physiol., in press. [[PubMed]
13. Béguin P., Crambert, G., Guennoun, S., Garty, H., Horisberger, J.-D. & Geering, K. (2001) EMBO J.20, 3993-4002.
14. Béguin P., Wang, X. Y., Firsov, D., Puoti, A., Claeys, D., Horisberger, J. D. & Geering, K. (1997) EMBO J.16, 4250-4260.
15. Arystarkhova E., Donnet, C., Asinovski, N. K. & Sweadner, K. J. (2002) J. Biol. Chem.277, 10162-10172. [[PubMed]
16. Pu H. X., Cluzeaud, F., Goldshlegger, R., Karlish, S. J. D., Farman, N. & Blostein, R. (2001) J. Biol. Chem.276, 20370-20378. [[PubMed]
17. Morrison B. W., Moorman, J. R., Kowdley, G. C., Kobayashi, Y. M., Jones, L. R. & Leder, P. (1995) J. Biol. Chem.270, 2176-2182. [[PubMed]
18. Fu X. & Kamps, M. (1997) Mol. Cell. Biol.17, 1503-1512.
19. Yamaguchi F., Yamaguchi, K., Tai, Y., Sugimoto, K. & Tokuda, M. (2001) Brain Res. Mol. Brain Res.86, 189-192. [[PubMed]
20. Palmer C. J., Scott, B. T. & Jones, L. R. (1991) J. Biol. Chem.266, 11126-11130. [[PubMed]
21. Chen L. S. K., Lo, C. F., Numann, R. & Cuddy, M. (1997) Genomics41, 435-443. [[PubMed]
22. Moorman J. R., Ackerman, S. J., Kowdley, G. C., Griffin, M. P., Mounsey, J. P., Chen, Z. H., Cala, S. E., Obrian, J. J., Szabo, G. & Jones, L. R. (1995) Nature (London)377, 737-740. [[PubMed]
23. Kowdley G. C., Ackerman, S. J., Chen, Z. H., Szabo, G., Jones, L. R. & Moorman, J. R. (1997) Biophys. J.72, 141-145.
24. Mahmmoud Y. A., Vorum, H. & Cornelius, F. (2000) J. Biol. Chem.275, 35969-35977. [[PubMed]
25. Jewell E. A. & Lingrel, J. B. (1991) J. Biol. Chem.266, 16925-16930. [[PubMed]
26. Melton D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. & Green, M. R. (1984) Nucleic Acids Res.12, 7035-7056.
27. Geering K., Beggah, A., Good, P., Girardet, S., Roy, S., Schaer, D. & Jaunin, P. (1996) J. Cell Biol.133, 1193-1204.
28. Jones L. R. (1988) Methods Enzymol.157, 85-91. [[PubMed]
29. Crambert, G., Béguin, P., Pestov, N. B., Modyanov, N. N. & Geering, K. (2002) Biochemistry, in press.
30. Pressley T. A. (1992) Am. J. Physiol.262, C743-C751. [[PubMed]
31. Jaisser F., Jaunin, P., Geering, K., Rossier, B. C. & Horisberger, J. D. (1994) J. Gen. Physiol.103, 605-623.
32. Hasler U., Wang, X., Crambert, G., Béguin, P., Jaisser, F., Horisberger, J. D. & Geering, K. (1998) J. Biol. Chem.273, 30826-30835. [[PubMed]
33. Wang J. N., Schwinger, R. H. G., Frank, K., Mullerehmsen, J., Martin-Vasallo, P., Pressley, T. A., Xiang, A., Erdmann, E. & McDonough, A. A. (1996) J. Clin. Invest.98, 1650-1658.
34. Thompson C. & McDonough, A. (1996) J. Biol. Chem.271, 32653-32658. [[PubMed]
35. Therien A. G., Goldshleger, R., Karlish, S. J. & Blostein, R. (1997) J. Biol. Chem.272, 32628-32634. [[PubMed]
36. Arystarkhova E., Wetzel, R. K., Asinovski, N. K. & Sweadner, K. J. (1999) J. Biol. Chem.274, 33183-33185. [[PubMed]
37. Walaas S. I., Czernik, A. J., Olstad, O. K., Sletten, K. & Walaas, O. (1994) Biochem. J.304, 635-640.
38. Moorman J. R., Palmer, C. J., John, J. E., III, Durieux, M. E. & Jones, L. R. (1992) J. Biol. Chem.267, 14551-14554. [[PubMed]
39. Morales-Mulia M., Pasantes-Morales, H. & Moran, J. (2000) Biochim. Biophys. Acta1496, 252-260. [[PubMed]
40. Moran J., Morales-Mulia, M. & Pasantes-Morales, H. (2001) Biochim. Biophys. Acta1538, 313-320. [[PubMed]
41. Glitsch H. G. (2001) Physiol. Rev.81, 1791-1826. [[PubMed]
42. Barlet-Bas C., Cheval, L., Khadouri, C., Marsy, S. & Doucet, A. (1990) Am. J. Physiol.259, F246-F250. [[PubMed]
43. Mihailidou A. S., Bundgaard, H., Mardini, M., Hansen, P. S., Kjeldsen, K. & Rasmussen, H. H. (2000) Circ. Res.86, 37-42. [[PubMed]
44. Béguin P., Crambert, G., Monnet-Tschudi, F., Uldry, M., Horisberger, J.-D., Garty, H. & Gerring, K. (2002) EMBO J.21, 3264-3273.