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

Abstract

Rapid and precise control of Na؉ /Ca 2؉ exchanger (NCX1) activity is essential in the maintenance of beatto-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.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

“…Reverse mode NCX1 was also observed to be inhibited in Ca 2ϩ fluorescence measurements. This result is in agreement with Ahlers et al (22), who used a radioactive tracer uptake method in HEK293 cells. They found FIGURE 11.…”

“…Co-expression of PLM with WT NCX1(FL) resulted in NCX1 inhibition (both forward and reverse mode), consistent with what was reported previously (Fig. 7F) (PLM is endogenously phosphorylated 30 -40% by kinases in HEK293) (22). Moreover, mutation of the PP1c-anchoring site in NCX1 (NCX1(F407P)) resulted in more NCX1 inhibition (Fig.…”

“…PP1c Anchored to NCX1-KIFF/KVFF, Dephosphorylates pSer-68-PLM, and Regulates NCX1 Activity in HEK293-We confirmed that PLM co-precipitates with NCX1 in lysates from rat cardiomyocytes and HEK293, as reported previously (22). Immunoblotting with a specific anti-pSer-68-PLM antibody showed that PLM was highly phosphorylated in HEK293, consistent with reports that PLM is endogenously phosphorylated (30 -40%) by kinases in HEK293 (24,47).…”

“…Immunoblotting with a specific anti-pSer-68-PLM antibody showed that PLM was highly phosphorylated in HEK293, consistent with reports that PLM is endogenously phosphorylated (30 -40%) by kinases in HEK293 (24,47). Noteworthy, both PLM and non-phosphorylatable PLM bind to NCX1 (22); however, it is pSer-68-PLM that exerts an inhibitory effect on NCX1 activity (24). The pSer-68-PLM level was significantly reduced in the presence of NCX1(FL)-GFP, suggesting that NCX1 anchoring is a prerequisite for dephosphorylation of pSer-68-PLM by PP1c.…”

“…Accumulating data indicate that PLM is one of these regulatory players. This 72-amino acid transmembrane protein, belonging to the FXYD1 family of ion transporters (20), co-localizes and coimmunoprecipitates with NCX1 and has been shown to inhibit NCX1 activity when it is phosphorylated at serine 68 (pSer-68-PLM) (21)(22)(23)(24). Interestingly, pSer-68-PLM relieves inhibition of the Na ϩ /K ϩ -ATPase (NKA), causing an increase in NKA activity (25,26), suggesting that PLM may serve as a regulator of both NCX (24) and NKA (27) depending on its phosphorylation status.…”