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J. Biol. Chem., Vol. 281, Issue 42, 32004-32014, October 20, 2006

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Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na⁺/Ca²⁺ Exchanger^*

JuFang Wang¹, Xue-Qian Zhang¹, Belinda A. Ahlers, Lois L. Carl, Jianliang Song, Lawrence I. Rothblum, Richard C. Stahl, David J. Carey, and Joseph Y. Cheung^¶2

From the Department of Cellular and Molecular Physiology and ^¶Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033 and the Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania 17822

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.

Received for publication, July 19, 2006 , and in revised form, August 17, 2006.

^* This work was supported in part by National Institutes of Health Grants HL-58672 and HL-74854 (to J. Y. C.), DK-46678 (to J. Y. C.), GM-69841 and HL-77814 (to L. I. R.), NS-21925 and NS-41363 (to D. J. C.); by American Heart Association Pennsylvania Affiliate Grants-in-Aid 0265426U (to X.-Q. Z.) and 0355744U (to J. Y. C.); by American Heart Association Pennsylvania Affiliate Post-Doctoral Fellowship 0425319U (to B. A. A.); and by grants from the Geisinger Foundation (to L. I. R. and D. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ J. Wang and X.-Q. Zhang contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, MC-H166, Hershey, PA 17033. Tel.: 717-531-5748; Fax: 717-531-7667; E-mail: jyc1{at}psu.edu.

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