Na+-K+-ATPase (NKA) establishes the transmembrane [Na+] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na+ affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM–NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP–NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT–PLM–YFP in HEK293 cells. NKA–PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA–PLM FRET. In contrast, NKA–PLM coimmunoprecipitation was only slightly reduced by E960A–NKA or F28A–PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960–NKA and F28–PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na+transport in intact cells. For WT–NKA, PLM reduced apparent Na+affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A–NKA the apparent Na+-affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA–PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Dec 11 2012|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS. We thank Khanha Dao, Jeffrey H. Elliott, and Garrick K. Yuen for technical help; Dr. Kenneth S. Ginsburg for comments on the manuscript; and Dr. Seth Robia for valuable intellectual contributions. This work was supported by National Institutes of Health (NIH) Grant R01-HL81562 (to D.M.B.). Molecular graphics images were produced using the chimera package from Resource for Biocomputing, Visualization, and Informatics at the University of California at San Francisco (supported by NIH P41 RR001081).
© 2012, National Academy of Sciences. All rights reserved.
ASJC Scopus subject areas