Identification of an endogenous inhibitor of the cardiac Na+/Ca2+ exchanger, phospholemman.
Rapid and precise control of Na(+)/Ca(2+) exchanger (NCX1) activity is essential in the maintenance of beat-to-beat Ca(2+) homeostasis in cardiac myocytes. Here, we show that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, is a novel endogenous protein inhibitor of cardiac NCX1. Using a heterologous expression system that is devoid of both endogenous PLM and NCX1, we first demonstrated by confocal immunofluorescence studies that both exogenous PLM and NCX1 co-localized at the plasma membrane. Reciprocal co-immunoprecipitation studies revealed specific protein-protein interaction between PLM and NCX1. The functional consequences of direct association of PLM with NCX1 was the inhibition of NCX1 activity, as demonstrated by whole-cell patch clamp studies to measure NCX1 current density and radiotracer flux assays to assess Na(+)-dependent (45)Ca(2+) uptake. Inhibition of NCX1 by PLM was specific, because a single mutation of serine 68 to alanine in PLM resulted in a complete loss of inhibition of NCX1 current, although association of the PLM mutant with NCX1 was unaltered. In native adult cardiac myocytes, PLM co-immunoprecipitated with NCX1. We conclude that PLM, a member of the FXYD family of small ion transport regulators known to modulate Na(+)-K(+)-ATPase, also regulates Na(+)/Ca(2+) exchange in the heart.
Ahlers
Belinda A
BA
Department of Cellular and Molecular Physiology, Pennsylvania State University, Hershey, 17033, USA.
Zhang
Xue-Qian
XQ
Moorman
J Randall
JR
Rothblum
Lawrence I
LI
Carl
Lois L
LL
Song
Jianliang
J
Wang
Jufang
J
Geddis
Lisa M
LM
Tucker
Amy L
AL
Mounsey
J Paul
JP
Cheung
Joseph Y
JY
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; C069141:phospholemman; D004247:DNA