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Background: We have previously shown that poly-L-lysine (L-Lys) to a significant extent prevents the inhibitory effect of B on PP. This phenomenon must depend on binding of B to the polypeptide (P), and might depend on the steric conformation of lysine. Methods: Phospholemman[50 μM]was incubated at 30°C, pH 7.4 for 3 min in microtiter plates containing (final conc.): 0.1 μM BSA, 0.2 mM ATP (with 1-4 μCi[γ-32P]ATP per assay), and 2.5 nM PKA. Reactions were performed± B [50 μM] and either L-Lys or poly-D-lysine (D-Lys) [300 μM], and were stopped by adding 0.2 M EDTA. The solution was transferred to SpinZyme separation units and washed ×3 with 75 mM phosphoric acid. Radioactivity incorporated in the substrate was measured as Cerenkov radiation. Results: Both L-Lys and D-Lys significantly reduced the inhibitory effect of B on PP (see figure). The contrasts between +B/-P and +B/+L-Lys or +B/-P and +B/+D-Lys were both highly statistically significant (Mann-Whitney U' = 49, p=0.0006 for both). The contrast between +B/+L-Lys and +B/ +D-Lys was not significant (Mann-Whitney U=17, p=0.38). Discussion: Our previous finding that L-Lys significantly reduced the inhibitory effect of B on PP, as well as our finding in a companion abstract that a lysine-containing dekapeptide which mimics subdomain II on protein kinases, also reduced the inhibitory effect of B on PP, both support the hypothesis that binding of B to lysine may be important in the mediation or modulation of B toxicity. Here we have shown that changing the steric conformation of lysine does not alter its ability to modify B toxicity. This provides further insight into the mechanisms mediating and/or modifying the effects of B on cell regulatory mechanisms.

A LYSINE(Lys)-CONTAINING PEPTIDE CORRESPONDING TO A TEN AMINOACID SEQUENCE OF PROTEIN KINASE SUBDOMAIN II PARTIALLY PREVENTS THE INHIBITORY EFFECT OF BILIRUBIN (B) ON PHOSPHOLEMMAN PHOSPHORYLATION (P) BY cAMP-DEPE

The effects of alpha- and beta-adrenergic stimulation on sarcolemmal protein phosphorylation and contractile slow responses were studied in intact myocardium. Isolated rat ventricles were perfused via the coronary arteries with 32Pi after which membrane vesicles partially enriched in sarcolemma were isolated from individual hearts. Alterations in the sarcolemmal slow inward Ca2+ current were assessed in the 32P-perfused hearts using a contractile slow response model. In this model, Na+ channels were first inactivated by partial depolarization of the hearts in 25 mM K+ after which alterations in Ca2+ channel activity produced by either alpha- or beta-adrenergic agonists could be assessed as restoration of contractions. alpha-Adrenergic stimulation (phenylephrine + propranolol) of the perfused hearts resulted in increased 32P incorporation into a 15-kDa sarcolemmal protein. This protein co-migrated with the 15-kDa sarcolemmal protein phosphorylated in hearts exposed to beta-adrenergic stimulation produced by isoproterenol. beta-Adrenergic stimulation, but not alpha-adrenergic stimulation, also resulted in phosphorylation of the sarcoplasmic reticulum protein, phospholamban. Phosphorylation of the 15-kDa protein in perfused hearts in response to either alpha- or beta-adrenergic stimulation was associated with restoration of contractions, indicative of increases in the slow inward Ca2+ current. Increases in 32P incorporation into the 15-kDa protein preceded restoration of contractions by phenylephrine. Nifedipine abolished the contractile responses to alpha-adrenergic stimulation while having no effect on increases in 15-kDa protein phosphorylation. The effects of alpha-adrenergic stimulation occurred in the absence of increases in cAMP levels. These results suggest that phosphorylation of the 15-kDa protein may be involved in increases in the slow inward current produced by stimulation of either alpha- or beta-adrenergic receptors.

The effect of beta-adrenergic stimulation on sarcolemmal protein phosphorylation was examined in intact ventricular myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with 32Pi after which membrane vesicles enriched 3-5-fold in sarcolemma were isolated by differential centrifugation followed by sucrose gradient centrifugation. Perfusion of hearts with isoproterenol stimulated 32P incorporation into a protein of apparent molecular weight of 15,000, which copurified with sarcolemmal vesicles. The increase in 32P incorporation was rapid in onset and elevated 2.5-3.0-fold after 30-45 s exposure of hearts to 100 nM isoproterenol. A positive correlation was found between stimulation of phosphorylation of the 15-kDa protein and the increase in the maximal rate of developed tension in intact ventricles after administration of isoproterenol. Phosphorylated phospholamban (most likely present as a contaminant) was also identified in the same sarcolemmal preparations. However, phospholamban and the 15-kDa sarcolemmal substrate were different proteins. Boiling of the membrane samples in sodium dodecyl sulfate prior to electrophoresis dissociated the high Mr form of phospholamban into the form of lower Mr but did not alter the mobility of the 15-kDa protein in sodium dodecyl sulfate-polyacrylamide gels. The 15-kDa protein did not undergo the electrophoretic mobility shift that is characteristic of phospholamban after cAMP-dependent phosphorylation nor did it cross-react with a highly specific phospholamban antibody. In vitro phosphorylation experiments conducted with the unmasking agent Triton X-100 suggested that the 15-kDa protein was localized to the cytoplasmic surfaces of sarcolemmal vesicles. These results demonstrate phosphorylation of a sarcolemmal protein, distinct from phospholamban, in response to beta-adrenergic stimulation of the heart. Phosphorylation of the sarcolemmal 15-kDa protein may play a role in mediating the effects of beta-adrenergic agonists on cardiac contractile force.

Phospholemman (PLM), a 72-amino acid membrane protein with a single transmembrane domain, forms taurine-selective ion channels in lipid bilayers. Because taurine forms zwitterions, a taurine-selective channel might have binding sites for both anions and cations. Here we show that PLM channels indeed allow fluxes of both cations and anions, making instantaneous and voltage-dependent transitions among conformations with drastically different ion selectivity characteristics. This surprising and novel ion channel behavior offers a molecular explanation for selective taurine flux across cell membranes and may explain why molecules in the phospholemman family can induce cation- or anion-selective conductances when expressed in Xenopus oocytes.

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.

The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [ 3 H]taurine and 125 I efflux induced by hyposmolarity were markedly increased (30–100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50–60%) of [ 3 H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [ 3 H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.

As the only quantitatively significant Na efflux pathway from cardiac cells, the Na/K ATPase (Na pump) is the primary regulator of intracellular Na. The transmembrane Na gradient it establishes is essential for normal electrical excitability, numerous coupled-transport processes and, as the driving force for Na/Ca exchange, thus setting cardiac Ca load and contractility. As Na influx varies with electrical excitation, heart rate and pathology, the dynamic regulation of Na efflux is essential. It is now widely recognized that phospholemman, a 72 amino acid accessory protein which forms part of the Na pump complex, is the key nexus linking cellular signaling to pump regulation. Phospholemman is the target of a variety of post-translational modifications (including phosphorylation, palmitoylation and glutathionation) and these can dynamically alter the activity of the Na pump. This review summarizes our current understanding of the multiple regulatory mechanisms that converge on phospholemman and govern NA pump activity in the heart. The corrected Fig. 4 is reproduced below. The publisher would like to apologize for any inconvenience caused. [corrected].

Treatment of bovine pulmonary smooth muscle cells with U46619 inhibited the Na(+)/K(+) ATPase activity in two parallel pathways: one of which is mediated via glutathionylation of the pump and the other by augmenting the inhibitory activity of the 70kDa inhibitor protein of Na(+)/K(+) ATPase. Although phospholemman deglutathionylates the pump leading to its activation, the inhibitor is responsible for irreversible inhibition of Na(+)/K(+) ATPase in an isoform specific manner during treatment of the cells with U46619.

A new type of chloride channel has been identified by functional expression of phospholemman, a 72-amino acid cardiac sarcolemmal protein with a single transmembrane domain. Xenopus oocytes injected with phospholemman RNA developed a chloride-selective current, which was activated by hyperpolarizing pulses. The current activated very slowly with a pronounced sigmoidal delay, did not inactivate, and increased in amplitude with trains of pulses, depolarized holding potentials, and low extracellular pH. Point mutations within the single transmembrane region abolished the sigmoidal delay of expressed currents. Phospholemman appears to be the smallest plasma membrane channel protein yet known. The structure is dissimilar to any chloride channel described thus far.

Les protéines FXYD appartiennent à une famille de petites protéines membranaires. Des études récentes suggèrent que six des sept membres de cette famille, FXYD1 (phospholemman), FXYD2 (sous-unité γ de la Na,K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (Ric) et FXYD7, sont des sous-unités auxiliaires de la Na, K-ATPase régulant son activité de manière tissu et isoforme spécifique. Ces résultats soulignent la complexité de la régulation des ions Na+ et K+ par la Na,K-ATPase qui est nécessaire pour assurer les fonctions propres de différents tissus comme la réabsorption du Na+ par le rein, la contraction musculaire et l’excitabilité neuronale. De plus, une mutation dans FXYD2 a été liée à certains cas d’hypomagnésémie, suggérant que des perturbations de la régulation de la Na,K-ATPase par les protéines FXYD seraient impliquées dans des états physiopathologiques. Une meilleure compréhension de ce nouveau mécanisme de régulation de la Na,K-ATPase pourrait nous aider à mieux comprendre son rôle dans les états physiopathologiques. Dans cet article, nous discutons les données les plus récentes sur le rôle des protéines FXYD dans la modulation de la Na, K-ATPase., Members of the FXYD protein family are small membrane proteins which are characterized by an FXYD motif, two conserved glycines and a serine residue. FXYD proteins show a tissue-specific distribution. Recent evidence suggests that 6 out of 7 FXYD proteins, FXYD1 (phospholemman), FXYD2 (γ subunit of Na,K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), FXYD5 (Ric) and FXYD7 associate with Na,K-ATPase and modulate its transport properties e.g. its Na+ and/or its K+ affinity in a distinct way. These results highlight the complex regulation of Na+ and K+ transport which is necessary to ensure proper tissue functions such as renal Na+-reabsorption, muscle contractility and neuronal excitability. Moreover, mutation of a conserved glycine residue into an arginine residue in FXYD2 has been linked to cases of human hypomagnesemia indicating that dysregulation of Na,K-ATPase by FXYD proteins may be implicated in pathophysiological states. A better characterization of this novel regulatory mechanism of Na,K-ATPase may help to better understand its role in physiological and pathophysiological conditions.

FXYD1 is a transmembrane protein predominantly expressed in excitable tissues that associates with and regulates Na/K ATPase. PKA phosphorylates FXYD1 at serine 68 (S68), however, the effects of phosphorylation on Na/K ATPase activity are not fully characterized. The objectives of this study were to characterize Na/K ATPase currents in FXYD1 wild-type (WT) and knockout (KO) adult mouse ventricular myocytes, and investigate the effects of FXYD1 on Na/K ATPase currents using the whole-cell patch-clamp technique. A peptide representing the 19 C-terminal residues of FXYD1 (FXYD1(54-72)) was introduced into the interior of FXYD1 KO and WT myocytes through the patch pipette. K-sensitive Na/K ATPase currents were higher in KO myocytes (2.9+/-0.1 pA/pF; n=4) compared with WT (1.9+/-0.1 pA/pF; n=4). Unphosphorylated FXYD1(54-72), at a concentration of 4 microM, reduced the currents in WT (from 2.1+/-0.1 to 1.3+/-0.1 pA/pF; P

We investigate the potential of multiple quantum filtered (MQF) 23Na NMR to probe intracellular [Na]i in the Langendorff perfused mouse heart. In the presence of Tm(DOTP) shift reagent the triple quantum filtered (TQF) signal originated largely from the intracellular sodium pool with a 32 ± 6% contribution of the total TQF signal arising from extracellular sodium, whilst the rank 2 double-quantum filtered signal (DQF), acquired with a 54.7° flip-angle pulse, originated exclusively from the extracellular sodium pool. Given the different cellular origins of the 23Na MQF signals we propose that the TQF/DQF ratio can be used as a semi-quantitative measure of [Na]i in the mouse heart. We demonstrate a good correlation of this ratio with [Na]i measured with shift reagent at baseline and under conditions of elevated [Na]i. We compare the measurements of [Na]i using both shift reagent and TQF/DQF ratio in a cohort of wild type mouse hearts and in a transgenic PLM3SA mouse expressing a non-phosphorylatable form of phospholemman, showing a modest but measurable elevation of baseline [Na]i. MQF filtered 23Na NMR is a potentially useful tool for studying normal and pathophysiological changes in [Na]i, particularly in transgenic mouse models with altered Na regulation.

The cardiac sodium/calcium exchanger (NCX1) regulates intracellular Ca in cardiac muscle. Inappropriate NCX1 function contributes to cardiac contraction abnormalities and heart failure: reduced NCX1 activity reduces Ca removal therefore impairing relaxation, whereas overactive NCX1 unloads the intracellular Ca stores and impairs systolic function. Although the structure-function relationship of NCX1 is well characterised, dynamic regulation of NCX1 function by post-translational modifications is controversial. The reversible attachment of the 16-carbon fatty acid palmitate to cysteine residues in proteins (palmitoylation) is an important and common post-translational modification in a variety of tissues. The palmitoyl proteome in cardiac tissue remains largely uncharacterised. Few proteins have been identified as palmitoylated, for example the sodium pump regulator phospholemman. We investigated the palmitoylation of NCX1 from isolated adult rat ventricular myocytes (ARVM), using site-specific resin assisted capture (Acyl Rac). Acyl Rac purified 100% of the constitutively palmitoylated protein caveolin 3 from ARVM lysates, and 70% of NCX1, indicating a biologically meaningful fraction of NCX1 is palmitoylated in ventricular muscle. We transiently expressed NCX1 in HEK-293 cells and measured palmitoylation by Acyl Rac and cell surface localization using membrane impermeable biotinylation reagents. The palmitoyl acyl transferase inhibitor 2-bromopalmitate (2-BP) significantly reduced both NCX1 palmitoylation and cell surface expression to 51±11% of expression in untreated cells, p

Phospholemman (PLM), a FXYD family member, critically modulates Na pump (NKA) and thus is important in Na and Ca regulation in cardiac myocytes. PLM inhibits NKA by reducing its apparent Na affinity, effect relieved by PLM phosphorylation. However, the sites responsible for the NKA-PLM interaction are unknown. Based on the recent NKA crystal structure with the associated FXYDs, we constructed single-site CFP-NKAα1 mutants with alanine substitution at F956, E960, L964 and F967 sites. Mutated CFP-NKAα1 and wild-type (WT) PLM-YFP were co-expressed in HEK cells and their interaction was assessed using fluorescence resonance energy transfer (FRET). FRET efficiency was determined as the relative increase in donor fluorescence (ΔFCFP) upon acceptor photobleaching. The NKA-PLM FRET was not altered by the F956A and F967A mutations, was significantly reduced for L964A (ΔFCFP =6±1% vs. 17.9±2% for WT-NKA) and was abolished for E960A (ΔFCFP =0.1±1%). Alanine mutation of the PLM site F48, shown by X-ray crystallography to be within interaction distance of NKA E960, eliminated the FRET with WT NKA (ΔFCFP=0.2±4%). Mutation of the same site to cysteine, as in some FXYD proteins, did not affect the NKA-PLM FRET (ΔFCFP=18.5±1.7%). To determine whether PLM affects the function of the E960A NKA mutant, we measured the NKA-mediated Na-extrusion as a function of [Na]i in a cell line that stably expresses E960A-NKA, with and without WT PLM. The apparent NKA-E960A affinity for [Na]i was similar in the absence (Kd=9.7±1.0 mM) and in the presence (Kd=8.9±1.7 mM) of WT-PLM and was not significantly affected by forskolin-induced PLM phosphorylation. In contrast, WT-PLM reduced the Na-affinity of WT-NKA (Kd increased from 9.3±1.6 to 11.5±1.9 mM) and PLM phosphorylation lowered the Kd to 9.8±1.8 mM. Thus, our results demonstrate that the E960-F48 interaction is critical for the PLM-NKA association.

Phospholemman (PLM or FXYD1) associates with and modulates Na pump (NKA) function in a manner similar to phospholamban (PLB) regulation of SERCA. That is, PLM inhibits NKA by reducing its [Na]i-affinity and PLM phosphorylation relieves this inhibition. We have also shown that PLM forms homo-oligomers (as does PLB) but it is unknown how PLM-PLM interactions occur or what functional role PLM oligomers have. Here we use site-directed mutagenesis and FRET to identify which PLM residues are critical to the PLM-NKA and PLM-PLM association, respectively. We hypothesize that mutations affecting PLM-PLM affinity will enhance PLM-NKA complexes and display stronger NKA inhibition, and vice versa. Based on crystal NKA-FXYD structure, the PLM sites G20, F28 & G31 were selected as potential NKA interaction sites and I26, I29, L30, L33&L36 as putative PLM interaction sites (analogous to leucine zippers thought to mediate PLB pentamerization). Alanine substitution of F28 eliminated PLM-NKA FRET (Fdonor increase by only 0.2±4% vs 18±2% for WT). Despite only minor effects on PLM-NKA interaction for G31A (Fdonor increase by 14±1%), its PLM-PLM FRET was significantly increased (Fdonor increase by 55±3% vs 37±5% for WT). Alanine substitution of I26, L30 & L33 all significantly reduced PLM-PLM FRET (Fdonor increase by 15±2%, 13±3% and 20±3% respectively vs 37±5% for WT) and opposing effects on PLM-NKA FRET were observed (e.g. Fdonor increases by 33±6% for L33A vs 18±2% for WT). We conclude that PLM homo-oligomers have a structural basis that parallels PLB (Leucine zipper). Moreover our hypothesis that altering the PLM affinity for NKA also changes the interaction of PLM with other PLM molecules (and vice versa) seems to be correct. Our data support the existence of a dynamic equilibrium of PLM oligomers and PLM-NKA that exerts functional regulation of NKA.

Phospholemman (FXYD1, PLM), the principal sarcolemmal substrate for protein kinases A and C in the heart, is a regulator of the cardiac sodium pump. We investigated proteins that interact with PLM in adult rat ventricular myocytes using bifunctional crosslinking reagents and co-immunoprecipitation.Digitonin-permeabilized ventricular myocytes were treated with the heterobifunctional crosslinking reagent sulfo-lc-smpt (distance between reactive groups 2nm, reactive towards amino and sulfhydryl amino acid side chains), and cell lysates immunoblotted for PLM. We found sulfo-lc-smpt quantitatively crosslinked PLM to a 20-25kDa protein (electrophoretic mobility of PLM 15kDa, electrophoretic mobility of crosslinked adduct 37kDa). Co-immunoprecipitation experiments indicated that the crosslinked adduct was PLM linked to the anti-oxidant protein peroxiredoxin 6 (prdx6). PLM phosphorylation at serine 68 (protein kinase A activation with 10μM forskolin) or at serines 63, 68 and threonine 69 (protein kinase C activation with 300nM PMA) had no effect on the ability of sulfo-lc-smpt to crosslink PLM to prdx6. Hydrogen peroxide treatment of ventricular myocytes (1-100μM) was also without effect on the binding of prdx6 to PLM.In conclusion, our data suggest a new role for PLM in the heart. As well as being responsible for kinase-mediated regulation of the cardiac sodium pump, it is responsible for recruitment of prdx6 to this ion transporter. Prdx6 catalyzes the reduction of hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides using glutathione. Given the well-established sensitivity of the alpha subunit of the sodium pump to cysteine oxidation, recruitment of prdx6 to the sodium pump by PLM may be important to maintain pump activity during periods of oxidative stress.