We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na ؉ /Ca 2؉ 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 wildtype 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 ؉ /Ca 2؉ exchange in the heart.Phospholemman (PLM), 2 a 72-amino acid membrane phosphoprotein with a single transmembrane domain (1), belongs to the FXYD gene family of small ion transport regulators (2). With the exception of the, all other known members of the FXYD gene family have at least one serine or threonine within the cytoplasmic tail (2), indicating potential phosphorylation sites. In particular, PLM (FXYD1) is the only FXYD family member to have a consensus sequence for phosphorylation by PKA (RRXS), PKC (RXXSXR), and NIMA (never in mitosis A) kinase (FRX(S/T)). Indeed PLM has been shown to be phosphorylated by PKA at serine 68 and PKC at both serine 63 and serine 68 (3).To date, PLM has been demonstrated to modulate ion fluxes through both the Na ϩ -K ϩ -ATPase (4 -8) and the cardiac Na ϩ /Ca 2ϩ exchanger (NCX1) (9 -11). Based on analogy of phospholamban inhibition of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2) (12) and experimental observation on the effects of PLMS (a 15-kDa homologue of PLM isolated from shark rectal glands) on shark Na ϩ -K ϩ -ATPase (13, 14), the current working hypothesis is that the Na ϩ pump is inhibited by unphosphorylated PLM. On phosphorylation of PLM, inhibition of Na ϩ -K ϩ -ATPase is relieved. This hypothesis has been given strong support by the observation that the V max of sarcolemmal Na ϩ -K ϩ -ATPase is increased 3-fold after acute cardiac ischemia in association with increased PLM phosphorylation by Ͼ300% (5). In a...

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.

Phospholemman (PLM) is a 72-amino-acid peptide with a single transmembrane domain, the expression of which induces chloride currents in Xenopus oocytes. It has remained unknown whether PLM is an ion channel or acts as a channel regulator. Here we show, by measuring unitary anion currents across planar phospholipid bilayers to which immunoaffinity-purified recombinant PLM was added, that it does indeed form ion channels. Excised patches of oocytes expressing PLM had similar currents. Of the ions tested, the sulphonic amino acid taurine was the most permeant, and expression of PLM increased fluxes of radiolabelled taurine in oocytes. Phospholemman is the smallest protein in cell membranes known to form an ion channel and the taurine selectivity suggests that it is involved in cell volume regulation.

Overexpression of phospholemman (PLM) in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca2+ concentration ([Ca2+]i) homeostasis and inhibited Na+/Ca2+ exchanger (NCX1). In addition, PLM coimmunoprecipitated and colocalized with NCX1 in cardiac myocyte lysates. In this study, we evaluated whether the cytoplasmic domain of PLM is crucial in mediating its effects on contractility, [Ca2+]i transients, and NCX1 activity. Canine PLM or its derived mutants were overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. Confocal immunofluorescence images using canine-specific PLM antibodies demonstrated that the exogenous PLM or its mutants were correctly targeted to sarcolemma, t-tubules, and intercalated discs, with little to none detected in intracellular compartments. Overexpression of canine PLM or its mutants did not affect expression of NCX1, sarco(endo)plasmic reticulum Ca2+-ATPase, Na+-K+-ATPase, and calsequestrin in adult rat myocytes. A COOH-terminal deletion mutant in which all four potential phosphorylation sites (Ser62, Ser63, Ser68, and Thr69) were deleted, a partial COOH-terminal deletion mutant in which Ser68 and Thr69 were deleted, and a mutant in which all four potential phosphorylation sites were changed to alanine all lost wild-type PLM's ability to modulate cardiac myocyte contractility. These observations suggest the importance of Ser68 or Thr69 in mediating PLM's effect on cardiac contractility. Focusing on Ser68, the Ser68 to Glu mutant was fully effective, the Ser63 to Ala (leaving Ser68 intact) mutant was partially effective, and the Ser68 to Ala mutant was completely ineffective in modulating cardiac contractility, [Ca2+]i transients, and NCX1 currents. Both the Ser63 to Ala and Ser68 to Ala mutants, as well as PLM, were able to coimmunoprecipitate NCX1. It is known that Ser68 in PLM is phosphorylated by both protein kinases A and C. We conclude that regulation of cardiac contractility, [Ca2+]i transients, and NCX1 activity by PLM is critically dependent on Ser68. We suggest that PLM phosphorylation at Ser68 may be involved in cAMP- and/or protein kinase C-dependent regulation of cardiac contractility.