Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain.
Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at Ser(68), PLM inhibits cardiac Na(+)/Ca(2+) exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or Ca(2+)-binding (residues 371-508) domains, of NCX1. In this study, we used intact Na(+)/Ca(2+) exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current (I(NaCa)). Deletion of residues 240-679, 265-373, 250-300, or 300-373 from WT NCX1 resulted in loss of inhibition of I(NaCa) by PLM. Inhibition of I(NaCa) by PLM was preserved when residues 229-237, 270-300, 328-330, or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of I(NaCa) by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, I(NaCa) measured in mutants lacking residues 238-270, 300-328, or 238-270 + 300-328 was not affected by PLM. Glutathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-371, 218-320, 218-270, 238-371, and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating that residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of I(NaCa) by PLM phosphorylation observed with WT NCX1 was absent in 250-300 deletion mutant but preserved in 229-237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328.
Zhang
Xue-Qian
XQ
Division of Nephrology, Thomas Jefferson Univ., 833 Chestnut St., Suite 700, Philadelphia, PA 19107, USA.
Wang
Jufang
J
Carl
Lois L
LL
Song
Jianliang
J
Ahlers
Belinda A
BA
Cheung
Joseph Y
JY
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D006801:Humans; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D012694:Serine