title: Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger.
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.
authors.affiliation: Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
authors.forename: JuFang
authors.initials: J
authors.lastname: Wang
authors.forename: Xue-Qian
authors.initials: XQ
authors.lastname: Zhang
authors.forename: Belinda A
authors.initials: BA
authors.lastname: Ahlers
authors.forename: Lois L
authors.initials: LL
authors.lastname: Carl
authors.forename: Jianliang
authors.initials: J
authors.lastname: Song
authors.forename: Lawrence I
authors.initials: LI
authors.lastname: Rothblum
authors.forename: Richard C
authors.initials: RC
authors.lastname: Stahl
authors.forename: David J
authors.initials: DJ
authors.lastname: Carey
authors.forename: Joseph Y
authors.initials: JY
authors.lastname: Cheung
mesh_terms: D000818:Animals; D003593:Cytoplasm / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000737:chemistry*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D005982:Glutathione Transferase