Positive correlation of gene expression between branchial FXYD proteins and Na+/K+-ATPase of euryhaline milkfish in response to hypoosmotic challenges.

FXYD proteins are crucial regulators of Na+/K+-ATPase (NKA), which plays an important role in ion exchange by providing the driving force for other ion-transporting systems in the osmoregulatory organs, including the gills. In milkfish (Chanos chanos), gill NKA has been widely investigated and found to alter its expression (both mRNA and protein) and activity in response to environmental salinity changes. However, the expression and roles of the regulatory proteins of NKA, the FXYD proteins, in milkfish gills upon salinity challenge is not yet clear. Hence, this study illustrated the potential roles of milkfish branchial FXYD proteins in modulating NKA expression via identification and tissue distributions of FXYD proteins, as well as the effects of salinity on expression of gill fxyd and nka mRNA. Six milkfish FXYD proteins (CcFXYD) were identified. In milkfish gill, gill-specific Ccfxyd11 was the predominant member, followed by Ccfxyd9 and Ccfxyd8. Upon hypoosmotic challenges, increases in gill Ccfxyd11, Ccfxyd8, Ccnka α1, and Ccnka ß1 mRNA as well as significantly positive correlations were observed. Moreover, after acute salinity changes, expression of gill Ccfxyd11 and Ccnka was found to change with ambient salinity, and significant positive correlations were also exhibited between Ccfxyd11 and Ccnka α1. Overall, these results revealed close relationships between CcFXYD11 and CcNKA α1 in milkfish gills, highlighting the potential roles of CcFXYD11 in osmoregulation.

Hypermagnesuria in Humans Following Acute Intravenous Administration of Digoxin.

BACKGROUND: Hypomagnesemia is a known predisposing condition for the appearance of digitalis toxicity. The detection of a genetic form of Mg urinary wasting with hypomagnesemia being caused by a mutation in the γ subunit (FXYD2) of the Na,K-ATPase, the pharmacological target of Digoxin, prompted us to investigate whether Digoxin administration increases urinary Mg excretion. METHODS: Two groups of subjects, with rapid atrial fibrillation, received intravenous Digoxin (n = 9) or verapamil (n = 8), for heart rate control. During the following 4 h, blood and urinary creatinine, sodium, potassium, calcium, and magnesium levels were determined, and fractional excretion (Fex) values for Na, K, Ca, and Mg were calculated. RESULTS: In the Digoxin group, at 60 min Fex Mg rose from 3.07 ± 1.21 to 7.58 ± 2.51% (an increase of 269 ± 107% of baseline, p < 0.001), and at 240 min to 6.05 ± 2.30% (204 ± 56% of baseline, p < 0.01). No significant change was observed for Fex Na, Fex K, and Fex Ca. A striking correlation was found between individual values of Fex Mg and serum Digoxin concentration (r = 0.678, p < 0.0001). No significant correlation was found between Fex Na or Fex K and serum Digoxin. A correlation of borderline significance was found between Fex Ca and serum Digoxin (r = 0.349, p = 0.073). CONCLUSIONS: The hypermagnesuric effect of acute Digoxin treatment is reminiscent of the effect of the missense mutation in FXYD2, which assumes that FXYD2 is a positive regulator of Na,K-ATPase in the distal convoluted tubule (DCT). The borderline calciuric effect of Digoxin may point to an additional site of action, more proximal to the DCT, that is, the thick ascending limb.

Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

KEY POINTS: Small transmembrane proteins such as FXYDs, which interact with Na+ ,K+ -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. ABSTRACT: Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na+ ,K+ -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony vertebrates (Euteleostomi). Diversification of SERCA regulators was much less extensive, indicating they operate under different evolutionary constraints. Finally, we found that FXYDs in extant vertebrates can be classified into 13 gene subfamilies, which do not always correspond to the established FXYD classification. We therefore propose a revised classification that is based on evolutionary history of FXYDs and that is consistent across vertebrate species. Collectively, our findings provide an improved framework for investigating the function of ion transport in health and disease.

Induced overexpression of phospholemman S68E mutant improves cardiac contractility and mortality after ischemia-reperfusion.

Phospholemman (PLM), when phosphorylated at Ser(68), inhibits cardiac Na+ / Ca2+ exchanger 1 (NCX1) and relieves its inhibition on Na+ -K+ -ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8-10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ∼35-75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+ -ATPase, α1- and α2-subunits of Na+ -K+ -ATPase, α1c-subunit of L-type Ca2+ channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ∼47% but the NCX1 current was depressed by ∼34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+ -K+ -ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+ concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+ contents, and [Ca(2+)]i transient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+ content, and [Ca2+]i transient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/dt in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/dt and -dP/dt and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.

A separate pool of cardiac phospholemman that does not regulate or associate with the sodium pump: multimers of phospholemman in ventricular muscle.

BACKGROUND: Phospholemman regulates the plasmalemmal sodium pump in excitable tissues. RESULTS: In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump. CONCLUSION: Phospholemman oligomers exist in cardiac muscle. SIGNIFICANCE: Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.