Identification of an Endogenous Inhibitor of the Cardiac Na+/Ca2+ Exchanger, Phospholemman

Belinda A Ahlers,Xue-Qian Zhang,J Randall Moorman,Lawrence I Rothblum,Lois L Carl,Jianliang Song,Jufang Wang,Lisa M Geddis,Amy L Tucker,J Paul Mounsey,Joseph Y Cheung

doi:10.1074/jbc.m414703200

Belinda A Ahlers, Xue-Qian Zhang + Show 9 more

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https://doi.org/10.1074/jbc.m414703200

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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.

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Excitation-contraction coupling in cardiac myocytes depends on the ability of regulatory proteins to maintain steady-state Ca2ϩ fluxes through each cycle of Ca2ϩ influx, intracellular Ca2ϩ transient buffering, and Ca2ϩ efflux [1]
Using both a monoclonal antibody directed against the intracellular loop of NCX1 (R3F1; Fig. 1B) and a polyclonal antibody directed against the cytoplasmic domain of PLM (C2Ab; Fig. 1C) [11], we demonstrated that both proteins were predominantly present at the plasma membrane in permeabilized PLM ϩ NCX1-co-transfected cells
These results suggest that both PLMand NCX1-transfected cDNAs underwent similar sorting and/or processing of the translated protein as observed for endogenous PLM and NCX1 in cardiac myocytes [14]

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Excitation-contraction coupling in cardiac myocytes depends on the ability of regulatory proteins to maintain steady-state Ca2ϩ fluxes through each cycle of Ca2ϩ influx, intracellular Ca2ϩ transient buffering, and Ca2ϩ efflux [1]. Confocal immunofluorescence and co-immunoprecipitation studies of transiently transfected HEK293 cells clearly showed co-localization and association of both exogenous PLM and NCX1 at the plasma membrane.

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