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pubmed:abstractText |
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.
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