Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation.
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.
Zhang
Xue-Qian
XQ
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033, USA.
Ahlers
Belinda A
BA
Tucker
Amy L
AL
Song
Jianliang
J
Wang
JuFang
J
Moorman
J Randall
JR
Mounsey
J Paul
JP
Carl
Lois L
LL
Rothblum
Lawrence I
LI
Cheung
Joseph Y
JY
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
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