Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

BACKGROUND: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFß superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known. RESULTS: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding. CONCLUSION: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-subunit in response to stress.

Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH(2)O with NaCl for 48 h or adaptation to 700 mosmol/kgH(2)O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser(68) (1-5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH(2)O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice.

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.

FXYD proteins as regulators of the Na,K-ATPase in the kidney.

The FXYD gene family has seven members in mammals and others in fish. Five of these (FXYD1, FXYD2, FXYD4, FXYD7, and PLMS from shark) have been shown to alter the activity of the Na,K-ATPase, as described by other papers in this volume. The gene structure of FXYD family members suggests assembly from protein domain modules and gene duplication. The gamma subunit is unique in the family for having alternative splice variants in the coding region and can be posttranslationally modified with different final consequences for enzyme properties. The nonoverlapping distribution of gamma and CHIF (FXYD4) in kidney helps to explain physiological differences in Na(+) affinity among nephron segments. We also detected phospholemman (FXYD1) in kidney. By immunofluorescence, it was found in extraglomerular mesangial cells (EM cells) of the juxtaglomerular apparatus and in the afferent arteriole. Contrary to many reports that only alpha1 and beta1 are expressed in the kidney, we found that alpha2 and beta2 are present, although not in any nephron segment. Both were detected in arterioles, and beta2 was found in the EM cells. In contrast, alpha1, beta1, and gamma were found in adjacent macula densa. Phospholemman, alpha2, and beta2 are proposed to have distinct roles in regulating the sodium pump in structures involved in tubuloglomerular feedback.

Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus.

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase gamma subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase alpha subunit isoforms (alpha1-alpha3) from cerebellum, indicating that the interaction is not specific to a particular alpha isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activity in vitro without effect on Na+ affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found.

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1.

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.

Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit.

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(b), with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(b) also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na(+) affinity, but modification was required for the effect on K(+) affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na(+) affinity was shown by the slower growth of gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.