title: Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation.
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.
authors.affiliation: Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
authors.forename: Xue-Qian
authors.initials: XQ
authors.lastname: Zhang
authors.forename: Belinda A
authors.initials: BA
authors.lastname: Ahlers
authors.forename: Amy L
authors.initials: AL
authors.lastname: Tucker
authors.forename: Jianliang
authors.initials: J
authors.lastname: Song
authors.forename: JuFang
authors.initials: J
authors.lastname: Wang
authors.forename: J Randall
authors.initials: JR
authors.lastname: Moorman
authors.forename: J Paul
authors.initials: JP
authors.lastname: Mounsey
authors.forename: Lois L
authors.initials: LL
authors.lastname: Carl
authors.forename: Lawrence I
authors.initials: LI
authors.lastname: Rothblum
authors.forename: Joseph Y
authors.initials: JY
authors.lastname: Cheung
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000818:Animals; D002155:Calsequestrin / Q000737:chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D005576:Colforsin / Q000737:chemistry / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007477:Ions; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D032342:Muscle Cells / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D014162:Transfection
chemical_list: D002155:Calsequestrin; D007477:Ions; D008565:Membrane Proteins; C499849:NCX1 protein, mouse; D010750:Phosphoproteins; D019831:Sodium-Calcium Exchanger; C069141:phospholemman; D005576:Colforsin; D018698:Glutamic Acid; D012694:Serine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine