Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation.
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Journal: 2006/May - Journal of Biological Chemistry
ISSN: 0021-9258
Abstract:
We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I(NaCa)) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I(NaCa), (ii) attenuation of the increase in I(NaCa) by PMA, and (iii) additional reduction in I(NaCa) in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of I(NaCa), whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on I(NaCa). Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I(NaCa) as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I(NaCa). The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, I(NaCa) was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in I(NaCa) was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I(NaCa) in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.
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J Biol Chem 281(12): 7784-7792
PMID: 16434394

Phospholemman inhibition of the cardiac Na<sup>+</sup>/Ca<sup>2+</sup> exchanger: Role of phosphorylation.

Department of Cellular and Molecular Physiology and
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033;
Weis Center for Research, Geisinger Medical Center, Danville, PA 17822; and
Department of Internal Medicine (Cardiovascular Division), University of Virginia Health Sciences Center, Charlottesville, VA 22908.
Address Correspondence To: Joseph Y. Cheung, M.D., Ph.D., Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, MC-H166, Hershey, PA 17033. Tel. (717)531-5748; Fax. (717)531-7667; Email: ude.usp@1cyj
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Abstract

We have previously demonstrated that phospholemman (PLM), a 15 kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na/Ca exchanger (NCX1). In addition, p rotein kinase A phosphorylates serine while protein kinase C phosphorylates both serine and serine of PLM. Using HEK293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (INaCa) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in INaCa; (ii) attenuation of the increase in INaCa by PMA; and (iii) additional reduction in INaCa in cells treated with forskolin. Mutating serine to alanine (S63A) preserved PLM’s sensitivity to forskolin in terms of suppression of INaCa, whereas mutating serine to alanine (S68A) abolished PLM’s inhibitory effect on INaCa. Mutating serine to glutamic acid (phosphomimetic) resulted in additional suppression of I NaCa as compared to wild-type PLM. These results suggest that PLM phosphorylated at serine inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knockout (KO) mice. When compared to wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, PMA-induced increase in INaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on INaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine, inhibits Na/Ca exchange in the heart.

Abbreviations: The abbreviations used are: ANOVA, analysis of variance; 8-Br-cAMP, 8-bromoadenosine 3′, 5′ cyclic monophosphate; [Ca]o, extracellular Ca concentration; Cm, whole cell membrane capacitance; CMV, cytomegalovirus; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; EGTA, ethylene glycol-bis-(β-aminoethyl ether)N,N,N’,N’-tetraacetic acid; Em, membrane potential; em., emission; ex., excitation; ENaCa, equilibrium potential for Na, Ca exchange current; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK, human embryonic kidney; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; INaCa, Na, Ca exchange current; KO, knock-out; MEM, minimal essential media; NCX1, Na, Ca exchanger; NIMA, never in mitosis A; PKA, protein kinase A; PKC, protein kinase C; PLM, phospholemman; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; SE, standard error; SERCA2, sarco(endo)plasmic reticulum Ca-ATPase; SR, sarcoplasmic reticulum; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; Vmax, maximum velocity; WT, wild-type
Abstract

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