Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger.

Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
Journal of Biological Chemistry (Impact Factor: 4.65). 11/2006; 281(42):32004-14. DOI: 10.1074/jbc.M606876200
Source: PubMed

ABSTRACT Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.
    Biochimica et Biophysica Acta 12/2010; 1808(4):1021-31. · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca(2+) ATPase (PMCA) and the plasma membrane Na(+)/Ca(2+) exchanger (NCX) are the two mechanisms responsible for Ca(2+) extrusion. The NCX has low Ca(2+) affinity but high capacity for Ca(2+) transport, whereas the PMCA has a high Ca(2+) affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca(2+), and the NCX the dynamic role of counteracting large cytosolic Ca(2+) variations (especially in excitable cells). This view of the roles of the two Ca(2+) extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca(2+) regulation (in the 100 nM range) and the Ca(2+) transients generated by cell stimulation (in the μM range).
    Cold Spring Harbor perspectives in biology 01/2011; 3(2). · 9.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.
    Journal of Biological Chemistry 04/2012; 287(25):21130-41. · 4.65 Impact Factor

Full-text (2 Sources)

Available from
May 30, 2014