Abstract 1528: Phospholemman Regulates Cardiac Na+/Ca2+ Exchanger: Interaction with Residues 238–270 and 300–328 of the Intracellular Loop
Phospholemman (PLM), an integral sarcolemmal phosphoprotein that belongs to the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). We have previously demonstrated that the cytoplasmic tail of PLM interacts with the intracellular loop (residues 218–764), but not the transmembrane domains (residues 1–217 and 765–938) of NCX1. In this study, we used NCX1 deletion mutants to map out the interaction sites with PLM. We demonstrated by Western blotting that wild-type NCX1 and its deletion mutants were expressed in transfected HEK293 cells. Co-transfection with PLM and NCX1 (or its deletion mutants) in HEK293 cells did not affect NCX1 (or its deletion mutants) expression. Co-expression of PLM with wild-type NCX1 inhibited NCX1 current (INaCa). Deletion of residues 240 – 679, 265–373, 250–300 or 300–373 from wild-type NCX1 resulted in loss of inhibition of INaCa by PLM. By contrast, inhibition of INaCa 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 INaCa by interacting with residues 238–270 and 300–328 of NCX1. Glutathione S-transferase (GST) pull-down assays confirmed that PLM bound to residues 218–764, 218–371, 218–320, 218–270, 238–371 and 300–373, but not to residues 250–300 and 371–508 of NCX1, indicating residues 218–270 and 300–373 physically associated with PLM. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238–270 and 300–328.