A gene family of small membrane proteins, represented by phospholemman and the gamma subunit of Na,K-ATPase, was defined and characterized by the analysis of more than 1000 related ESTs (expressed sequence tags). In addition to new and more complete cDNA sequence for known family members (including MAT-8, CHIF, and RIC), the findings included two new family members and new splicing variants. A large number of EST replicates made it possible to derive curated DNA sequence with higher confidence and accuracy than from the sequencing of individual clones. The family has a core motif of 35 invariant and conserved amino acids centered on a single transmembrane span. Features of each predicted protein product were compared, and tissue distributions were determined. The gene family was named FXYD (pronounced fix-id) in recognition of invariant amino acids in its signature motif. The abundant proteins are involved in the control of ion transport.

FXYD proteins belong to a family of small-membrane proteins. Recent experimental evidence suggests that at least five of the seven members of this family, FXYD1 (phospholemman), FXYD2 (gamma-subunit of Na-K-ATPase), FXYD3 (Mat-8), FXYD4 (CHIF), and FXYD7, are auxiliary subunits of Na-K-ATPase and regulate Na-K-ATPase activity in a tissue- and isoform-specific way. These results highlight the complexity of the regulation of Na+ and K+ handling by Na-K-ATPase, which is necessary to ensure appropriate tissue functions such as renal Na+ reabsorption, muscle contractility, and neuronal excitability. Moreover, a mutation in FXYD2 has been linked to cases of human hypomagnesemia, indicating that perturbations in the regulation of Na-K-ATPase by FXYD proteins may be critically involved in pathophysiological states. A better understanding of this novel regulatory mechanism of Na-K-ATPase should help in learning more about its role in pathophysiological states. This review summarizes the present knowledge of the role of FXYD proteins in the modulation of Na-K-ATPase as well as of other proteins, their regulation, and their structure-function relationship.

Cardiac sympathetic stimulation activates beta-adrenergic (beta-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess beta-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 micromol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8+/-1.4 to 13.6+/-1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5+/-1.8 to 10.5+/-1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2+/-1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9+/-0.9 versus 16.2+/-1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different alpha-beta isozymes of NKA after coexpression in Xenopus oocytes, and with alpha1-beta, and less efficiently with alpha2-beta isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of alpha1-beta1 and alpha2-beta1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder linked to heterozygous de novo mutations in the MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), which represses gene transcription by binding to 5-methylcytosine residues in symmetrically positioned CpG dinucleotides. Direct MeCP2 targets underlying RTT pathogenesis remain largely unknown. Here, we report that FXYD1, which encodes a transmembrane modulator of Na(+), K(+) -ATPase activity, is elevated in frontal cortex (FC) neurons of RTT patients and Mecp2-null mice. Increasing neuronal FXDY1 expression is sufficient to reduce dendritic arborization and spine formation, hallmarks of RTT neuropathology. Mecp2-null mouse cortical neurons have diminished Na(+),K(+)-ATPase activity, suggesting that aberrant FXYD1 expression contributes to abnormal neuronal activity in RTT. MeCP2 represses Fxyd1 transcription through direct interactions with sequences in the Fxyd1 promoter that are methylated in FC neurons. FXYD1 is therefore a MeCP2 target gene whose de-repression may directly contribute to RTT neuronal pathogenesis.

The recent description of virus-induced fish IFNs has raised questions about the evolution of this complex antiviral system. Identification of the receptor of the zebrafish virus-induced IFN (zIFN) was sought to help resolve these questions. We set up an experimental system to study the zIFN system in the course of a viral infection of zebrafish embryos. In this setting, zIFN was induced by viral infection, and we identified zIFN-dependent induced transcripts. Embryos quickly died from the infection, but zIFN overexpression increased their survival. We took advantage of this experimental system to perform in vivo loss and gain of function analysis of candidate receptors of the class II helical receptor family and identified zCRFB1 and zCRFB5 as the two subunits of the zebrafish IFN receptor. Based on the organization of the zIFN gene and the protein structure of the identified receptor components, the virus-induced fish IFNs appear as orthologs of mammalian IFN-lambda, specifying type III IFN as the ancestral antiviral system of vertebrates.

Regulation of the Na/K ATPase by protein kinases is model-specific. We have observed a profound activation of the sarcolemmal Na/K ATPase during cardiac ischemia, which is masked by an inhibitor of the enzyme in the cytosol. The aim of these studies was to characterize the pathways involved in this activation in the Langendorff-perfused rat heart. Na/K ATPase activity was determined by measuring ouabain-sensitive phosphate generation by cardiac homogenates at 37 degrees C. In isolated sarcolemma, ischemia (30 min) caused a substantial activation of the Na/K ATPase compared with aerobic controls, which was abolished by perfusing the heart with staurosporine or H89. However, the alpha1 subunit of the Na/K ATPase was not phosphorylated during ischemia. The sarcolemmal protein phospholemman (PLM) was found associated with the Na/K ATPase alpha1 and beta1 but not alpha2 subunits, and PLM increased its association with the catalytic subunit of PKA following ischemia. In vitro 14-3-3 binding assays indicated that PLM was phosphorylated following ischemia. These results indicate that the ischemia-induced activation of the Na/K ATPase is indirect, through phosphorylation of PLM, which is an integral part of the Na/K ATPase enzyme complex in the heart. The role of PLM is analogous to phospholamban in regulating the sarcoplasmic reticulum calcium ATPase.

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ 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 wild-type 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+/Ca2+ exchange in the heart.

BACKGROUND

Cardiac Na/K-ATPase (NKA) regulates intracellular Na ([Na](i)), which in turn affects intracellular Ca and thus contractility via Na/Ca exchange. Recent evidence shows that phosphorylation of the NKA-associated small transmembrane protein phospholemman (PLM) mediates beta-adrenergic-induced NKA stimulation.

RESULTS

Here, we tested whether PLM phosphorylation during beta-adrenergic activation limits the rise in [Na](i), Ca transient amplitude, and triggered arrhythmias in mouse ventricular myocytes. In myocytes from wild-type (WT) mice, [Na](i) increased on field stimulation at 2 Hz from 11.1+/-1.8 mmol/L to a plateau of 15.2+/-1.5 mmol/L. Isoproterenol induced a decrease in [Na](i) to 12.0+/-1.2 mmol/L. In PLM knockout (PLM-KO) mice in which beta-adrenergic stimulation does not activate NKA, [Na](i) also increased at 2 Hz (from 10.4+/-1.2 to 17.0+/-1.5 mmol/L) but was unaltered by isoproterenol. The PLM-mediated decrease in [Na](i) in WT mice could limit the isoproterenol-induced inotropic state. Indeed, the isoproterenol-induced increase in the amplitude of Ca transients was significantly smaller in the WT mice (5.2+/-0.4- versus 7.1+/-0.5-fold in PLM-KO mice). This also was the case for the sarcoplasmic reticulum Ca content, which increased by 1.27+/-0.09-fold in WT mice versus 1.53+/-0.09-fold in PLM-KO mice. The higher sarcoplasmic reticulum Ca content in PLM-KO versus WT mice was associated with an increased propensity for spontaneous Ca transients and contractions in PLM-KO mice.

CONCLUSIONS

These data suggest that PLM phosphorylation and NKA stimulation are an integral part of the sympathetic fight-or-flight response, tempering the rise in [Na](i) and cellular Ca loading and perhaps limiting Ca overload-induced arrhythmias.

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is approximately 30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 +/- 0.2 pA/pF in unstimulated cells to 2.9 +/- 0.1 pA/pF for PKA and 3.4 +/- 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump alpha(1)-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

Phospholemman, a transmembrane, 72 residue protein enriched in striated muscle and heart [Palmer, Scott and Jones (1991) J. Biol. Chem. 266, 11126-11130], is phosphorylated in response to insulin [Walaas, Horn and Walaas (1991) Biochim. Biophys. Acta 1094, 92-102]. The present study is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS63IRRLS68TRRR (in the single letter code) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with Km values of 6-7 microM for both enzymes. Amino acid sequencing of the phosphopeptide shows that protein kinase C phosphorylates both Ser-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates Ser-68. Thermolytic phosphopeptide mapping of 32P-labelled phospholemman from rat diaphragms shows that treatment with insulin results in labelling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide containing Ser-68. Hence, insulin and adrenaline regulate the phosphorylation of phospholemman, presumably through protein kinase C and cAMP-dependent protein kinase, respectively, on partly overlapping phosphorylation sites.

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving>>or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-alpha1 and NKA-alpha2 in a comparable but not identical manner.

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 Ca(2+)-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.

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

Because phospholemman (PLM) regulates the Na(+)/K(+) pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na(+)](i) decline (fluorescence measurements) and current (I(pump)) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, I(pump) (both at 10 and 100 mmol/L Na(+) in the pipette solution) and maximal NKA-mediated Na(+) extrusion rate (V(max)) from 7.9+/-1.1 to 12.7+/-1.9 mmol.L(-1) per minute without altering NKA affinity for internal Na(+) (K(0.5)). In PLM knockout mice, PDBu had no effect on either V(max) or K(0.5). After pretreatment with isoproterenol (ISO) (1 mumol/L), PDBu still increased the NKA V(max) and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na(+) affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly V(max) and K(0.5), respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.

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.

Defects in some of liver-enriched genes in mammals will cause liver- and/or blood-related diseases. However, due to the fact that embryogenesis happens intrauterinally in the mammals, the function of these liver-enriched genes during liver organogenesis is poorly studied. We report here the identification of 129 genuine liver-enriched genes in adult zebrafish and show that, through in situ hybridization, 69 of these genes are also enriched in the embryonic liver. External embryogenesis coupled with the well-established morpholino-mediated gene knock-down technique in zebrafish offers us a unique opportunity to study if this group of genes plays any role during liver organogenesis in the future. As an example, preliminary study using morpholino-mediated gene knock-down method revealed that a novel liver-enriched gene leg1 is crucial for the liver expansion growth. We also report the analysis of promoter regions of 51 liver-enriched genes by searching putative binding sites for Hnf1, Hnf3, Hnf4 and Hnf6, four key transcription factors enriched in the liver. We found that promoter regions of majority of liver-enriched genes contain putative binding sites for more than one HNF factors, suggesting that most of liver-enriched genes are likely co-regulated by different combination of HNF factors. This observation supports the hypothesis that these four liver-enriched transcription factors form a network in controlling the expression of liver-specific or -enriched genes in the liver.

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

mRNA transport and local translation in dendrites play key roles in use-dependent synaptic modification and in higher-order brain functions. RNG105, an RNA-binding protein, has previously been identified as a component of RNA granules that mediate dendritic mRNA localization and local translation. Here, we demonstrate that RNG105 knock-out in mice reduces the dendritic localization of mRNAs for Na+/K+ ATPase (NKA) subunit isoforms (i.e., α3, FXYD1, FXYD6, and FXYD7). The loss of dendritic mRNA localization is accompanied by the loss of function of NKA in dendrites without affecting the NKA function in the soma. Furthermore, we show that RNG105 deficiency affects the formation and maintenance of synapses and neuronal networks. These phenotypes are partly explained by an inhibition of NKA, which is known to influence synaptic functions as well as susceptibility to neurotoxicity. The present study first demonstrates the in vivo role of RNG105 in the dendritic localization of mRNAs and uncovers a novel link between dendritic mRNA localization and the development and maintenance of functional networks.

Previous studies showed increased phospholemman (PLM) mRNA after myocardial infarction (MI) in rats (Sehl PD, Tai JTN, Hillan KJ, Brown LA, Goddard A, Yang R, Jin H, and Lowe DG. Circulation 101: 1990-1999, 2000). We tested the hypothesis that, in normal adult rat cardiac myocytes, PLM overexpression alters contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. Compared with myocytes infected by control adenovirus expressing green fluorescent protein (GFP) alone, Western blots indicated a 41% increase in PLM expression after 72 h (P < 0.001) but no changes in Na(+)/Ca(2+) exchanger, SERCA2, and calsequestrin levels in myocytes infected by adenovirus expressing GFP and PLM. At 5 mM extracellular [Ca(2+)] ([Ca(2+)](o)), maximal contraction amplitudes in PLM-overexpressed myocytes were 24% (P < 0.005) and [Ca(2+)](i) transient amplitudes were 18% (P < 0.05) lower than control myocytes. At 0.6 mM [Ca(2+)](o), however, contraction and [Ca(2+)](i) transient amplitudes were significantly (P < 0.05) higher in PLM-overexpressed than control myocytes (18% and 42%, respectively); at 1.8 mM [Ca(2+)](o), the differences in contraction and [Ca(2+)](i) transient amplitudes were narrowed. This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-overexpressed myocytes mimics that observed in post-MI rat myocytes. We suggest that PLM overexpression observed in post-MI myocytes may partly account for contractile abnormalities by perturbing Ca(2+) fluxes during excitation-contraction.

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.