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Pulmonary hypertension (PH) consists of a heterogenous group of diseases that culminate in increased pulmonary arterial pressure and right ventricular (RV) dysfunction. We sought to investigate the role of FXYD1, a small membrane protein that modulates Na+-K+-ATPase function, in the pathophysiology of PH. We mined online transcriptome databases to assess FXYD1 expression in PH. We characterized the effects of FXYD1 knockout (KO) in mice on right and left ventricular (RV and LV) function using echocardiography and measured invasive hemodynamic measurements under normal conditions and after treatment with bleomycin sulfate or chronic hypoxia to induce PH. Using immunohistochemistry, immunoblotting, and functional assays, we examined the effects of FXYD1 KO on pulmonary microvasculature and RV and LV structure and assessed signaling via endothelial nitric oxide synthase (eNOS) and inflammatory pathways. FXYD1 lung expression tended to be lower in samples from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with controls, supporting a potential pathophysiological role. FXYD1 KO mice displayed characteristics of PH including significant increases in pulmonary arterial pressure, increased muscularization of small pulmonary arterioles, and impaired RV systolic function, in addition to LV systolic dysfunction. However, when PH was stimulated with standard models of lung injury-induced PH, there was no exacerbation of disease in FXYD1 KO mice. Both the lungs and left ventricles exhibited elevated nitrosative stress and inflammatory milieu. The absence of FXYD1 in mice results in LV inflammation and cardiopulmonary redox signaling changes that predispose to pathophysiological features of PH, suggesting FXYD1 may be protective. [ABSTRACT FROM AUTHOR]

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Na+/K+ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, β, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and β1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAβ1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K+ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min–1, p < 0.01) while also increasing cumulated leg K+ reuptake 0–3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAβ1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = –0.53, p < 0.05). NKAα2 and β1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAβ1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K+ shifts and enhance muscle performance during intense exercise. [ABSTRACT FROM AUTHOR]

Using bioinformatic approaches, we present evidence of distant relatedness among the Ephemerovirus Viroporin family, the Rhabdoviridae Putative Viroporin U5 family, the Phospholemman family, and the Small Integral Membrane Protein family. Our approach is based on the transitivity property of homology complemented with five validation criteria: (1) significant sequence similarity and alignment coverage, (2) compatibility of topology of transmembrane segments, (3) overlap of hydropathy profiles, (4) conservation of protein domains, and (5) conservation of sequence motifs. Our results indicate that Pfam protein domains PF02038 and PF15831 can be found in or projected onto members of all four families. In addition, we identified a 26-residue motif conserved across the superfamily. This motif is characterized by hydrophobic residues that help anchor the protein to the membrane and charged residues that constitute phosphorylation sites. In addition, all members of the four families with annotated function are either responsible for or affect the transport of ions into and/or out of the cell. Taken together, these results justify the creation of the novel Phospholemman/SIMP/Viroporin superfamily. Given that transport proteins can be found not just in cells, but also in viruses, the ability to relate viroporin protein families with their eukaryotic and bacterial counterparts is an important development in this superfamily. [ABSTRACT FROM AUTHOR]

In the heart, Na/K-ATPase regulates intracellular Na(+) and Ca(2+) (via NCX), thereby preventing Na(+) and Ca(2+) overload and arrhythmias. Here, we test the hypothesis that nitric oxide (NO) regulates cardiac intracellular Na(+) and Ca(2+) and investigate mechanisms and physiological consequences involved. Effects of both exogenous NO (via NO-donors) and endogenously synthesized NO (via field-stimulation of ventricular myocytes) were assessed in this study. Field stimulation of rat ventricular myocytes significantly increased endogenous NO (18 ± 2 μM), PKCε activation (82 ± 12%), phospholemman phosphorylation (at Ser-63 and Ser-68) and Na/K-ATPase activity (measured by DAF-FM dye, western-blotting and biochemical assay, respectively; p

In the heart, intracellular Na(+) concentration ([Na(+)]i) is a key modulator of Ca(2+) cycling, contractility and cardiac myocyte metabolism. Several Na(+) transporters are electrogenic, thus they both contribute to shaping the cardiac action potential and at the same time are affected by it. [Na(+)]i is controlled by the balance between Na(+) influx through various pathways, including the Na(+)/Ca(2+) exchanger and Na(+) channels, and Na(+) extrusion via the Na(+)/K(+)-ATPase. [Na(+)]i is elevated in HF due to a combination of increased entry through Na(+) channels and/or Na(+)/H(+) exchanger and reduced activity of the Na(+)/K(+)-ATPase. Here we review the major Na(+) transport pathways in cardiac myocytes and how they participate in regulating [Na(+)]i in normal and failing hearts. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes."

Background: Although it has long been recognized that smooth muscle Na/K ATPase modulates vascular tone and blood pressure (BP), the role of its accessory protein phospholemman has not been characterized. The aim of this study was to test the hypothesis that phospholemman phosphorylation regulates vascular tone in vitro and that this mechanism plays an important role in modulation of vascular function and BP in experimental models in vivo and in humans.Methods: In mouse studies, phospholemman knock-in mice (PLM3SA; phospholemman [FXYD1] in which the 3 phosphorylation sites on serines 63, 68, and 69 are mutated to alanines), in which phospholemman is rendered unphosphorylatable, were used to assess the role of phospholemman phosphorylation in vitro in aortic and mesenteric vessels using wire myography and membrane potential measurements. In vivo BP and regional blood flow were assessed using Doppler flow and telemetry in young (14-16 weeks) and old (57-60 weeks) wild-type and transgenic mice. In human studies, we searched human genomic databases for mutations in phospholemman in the region of the phosphorylation sites and performed analyses within 2 human data cohorts (UK Biobank and GoDARTS [Genetics of Diabetes Audit and Research in Tayside]) to assess the impact of an identified single nucleotide polymorphism on BP. This single nucleotide polymorphism was expressed in human embryonic kidney cells, and its effect on phospholemman phosphorylation was determined using Western blotting.Results: Phospholemman phosphorylation at Ser63 and Ser68 limited vascular constriction in response to phenylephrine. This effect was blocked by ouabain. Prevention of phospholemman phosphorylation in the PLM3SA mouse profoundly enhanced vascular responses to phenylephrine both in vitro and in vivo. In aging wild-type mice, phospholemman was hypophosphorylated, and this correlated with the development of aging-induced essential hypertension. In humans, we identified a nonsynonymous coding variant, single nucleotide polymorphism rs61753924, which causes the substitution R70C in phospholemman. In human embryonic kidney cells, the R70C mutation prevented phospholemman phosphorylation at Ser68. This variant's rare allele is significantly associated with increased BP in middle-aged men.Conclusions: These studies demonstrate the importance of phospholemman phosphorylation in the regulation of vascular tone and BP and suggest a novel mechanism, and therapeutic target, for aging-induced essential hypertension in humans. [ABSTRACT FROM AUTHOR]

Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by zDHHC5. Here, we show that PLM palmitoylation is facilitated by recruitment of the Na-pump α sub-unit to a specific site on zDHHC5 that contains a juxtamembrane amphipathic helix. Site-specific palmitoylation and GlcNAcylation of this helix increased binding between the Na-pump and zDHHC5, promoting PLM palmitoylation. In contrast, disruption of the zDHHC5-Na-pump interaction with a cell penetrating peptide reduced PLM palmitoylation. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by the acyltransferase zDHHC5. Plain & Howie et al identify a binding site on zDHHC5 for the Na-pump, discover that the interaction is regulated by post-translational modifications near this binding site, and find that disrupting the zDHHC5-Na-pump interaction reduces PLM palmitoylation. [ABSTRACT FROM AUTHOR]

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro , only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction. [Display omitted] • Identify peroxiredoxin 6 (Prdx6) as a thioesterase • Protein depalmitoylation by Prdx6 occurs via its catalytic cysteine • Phospholemman depalmitoylation by Prdx6 is glutathione dependent • Prdx6 thioesterase activity confers redox sensitivity on the Na pump The activities and cellular localizations of membrane proteins are frequently controlled by palmitoylation, a reversible covalent post-translational modification. Here, Howie et al. have shown that peroxiredoxin 6 is a novel thioesterase that removes palmitate from the acylated protein phospholemman in a glutathione-dependent mechanism, conferring redox sensitivity on Na pump activity. [ABSTRACT FROM AUTHOR]

Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM. [ABSTRACT FROM AUTHOR]

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the MECP2 gene. In the absence of MeCP2, expression of FXYD domain-containing transport regulator 1 (FXYD1) is deregulated in the frontal cortex (FC) of mice and humans. Because Fxyd1 is a membrane protein that controls cell excitability by modulating Na + , K + -ATPase activity (NKA), an excess of Fxyd1 may reduce NKA activity and contribute to the neuronal phenotype of Mecp2 deficient (KO) mice. To determine if Fxyd1 can rescue these RTT deficits, we studied the male progeny of Fxyd1 null males bred to heterozygous Mecp2 female mice. Maximal NKA enzymatic activity was not altered by the loss of MeCP2, but it increased in mice lacking one Fxyd1 allele, suggesting that NKA activity is under Fxyd1 inhibitory control. Deletion of one Fxyd1 allele also prevented the increased extracellular potassium (K + ) accumulation observed in cerebro-cortical neurons from Mecp2 KO animals in response to the NKA inhibitor ouabain, and rescued the loss of dendritic arborization observed in FC neurons of Mecp2 KO mice. These effects were gene-dose dependent, because the absence of Fxyd1 failed to rescue the MeCP2-dependent deficits, and mimicked the effect of MeCP2 deficiency in wild-type animals. These results indicate that excess of Fxyd1 in the absence of MeCP2 results in deregulation of endogenous K + conductances functionally associated with NKA and leads to stunted neuronal growth. [ABSTRACT FROM AUTHOR]

The ubiquitous sodium/potassium ATPase (Na pump) is the most abundant primary active transporter at the cell surface of multiple cell types, including ventricular myocytes in the heart. The activity of the Na pump establishes transmembrane ion gradients that control numerous events at the cell surface, positioning it as a key regulator of the contractile and metabolic state of the myocardium. Defects in Na pump activity and regulation elevate intracellular Na in cardiac muscle, playing a causal role in the development of cardiac hypertrophy, diastolic dysfunction, arrhythmias and heart failure. Palmitoylation is the reversible conjugation of the fatty acid palmitate to specific protein cysteine residues; all subunits of the cardiac Na pump are palmitoylated. Palmitoylation of the pump’s accessory subunit phospholemman (PLM) by the cell surface palmitoyl acyl transferase DHHC5 leads to pump inhibition, possibly by altering the relationship between the pump catalytic α subunit and specifically bound membrane lipids. In this review, we discuss the functional impact of PLM palmitoylation on the cardiac Na pump and the molecular basis of recognition of PLM by its palmitoylating enzyme DHHC5, as well as effects of palmitoylation on Na pump cell surface abundance in the cardiac muscle. We also highlight the numerous unanswered questions regarding the cellular control of this fundamentally important regulatory process. [ABSTRACT FROM AUTHOR]

The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes. [ABSTRACT FROM AUTHOR]

The pathogenesis of hypokalemic periodic paralysis (HypoPP) remains unclear. Though some mutations in skeletal muscle ion channels were revealed previously, the exact mechanism remains to be fully elucidated. Increased Na+/K+-ATPase activity in skeletal muscle is postulated to contribute to attacks of HypoPP. Before the link between Na+/K+-ATPase dysfunction and these ion channel mutations is established, mutations in Na+/K+-ATPase and their regulators are the first to be excluded. Phospholemman, which is a protein encoded by the FXYD domain-containing ion transport regulator 1 (FXYD1) gene, is predominantly expressed in skeletal muscle and is the major regulator of Na+/K+-ATPase. Therefore, the aim of the present study was to determine the genetic involvement of phospholemman in HypoPP development. Genomic DNA was extracted from the peripheral blood of five HypoPP probands with typical manifestations. The coding exons of FXYD1, exons 2-7, were polymerase chain reaction (PCR)-amplified and sequenced. No mutations were detected in FXYD1 in any of the subjects studied. To conclude, mutations in phospholemman encoding genes may not be involved with HypoPP and the relationship between phospholemman and Na+/K+-ATPase dysfunction in attacks of HypoPP requires further study. [ABSTRACT FROM AUTHOR]

FXYD proteins are important regulators of Na+/K+-ATPase (NKA) activity in mammals. As an inhabitant of estuaries, the pufferfish (Tetraodon nigroviridis) responds to ambient salinity changes with efficient osmoregulation, including alterations in branchial, and renal NKA activities. Previous studies on teleostean FXYDs have mainly focused on the expression and potential functions of FXYD proteins in gills. The goal of the present study was to elucidate the potential role of FXYD8, a member of the fish FXYD protein family, in the modulation of NKA activity in the kidneys of this euryhaline pufferfish by using molecular, biochemical, and physiological approaches. The results demonstrate that T. nigroviridis FXYD8 (TnFXYD8) interacts with NKA in renal tubules. Meanwhile, the protein expression of renal TnFXYD8 was found to be significantly upregulated in hyperosmotic seawater-acclimated pufferfish. Moreover, overexpression of TnFXYD8 in Xenopus oocytes decreased NKA activity. Our results suggest the FXYD8 is able to modulate NKA activity through inhibitory effects upon salinity challenge. The present study further extends our understanding of the functions of FXYD proteins, the regulators of NKA, in vertebrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

NCX1 (Na+ /Ca2+ exchanger 1) is an important regulator of intracellular Ca2+ and a potential therapeutic target for brain ischaemia and for diastolic heart failure with preserved ejection fraction. PLM (phospholemman), a substrate for protein kinases A and C, has been suggested to regulate NCX1 activity. However, although several studies have demonstrated that binding of phosphorylated PLM (pSer68-PLM) leads to NCX1 inhibition, other studies have failed to demonstrate a functional interaction of these proteins. In the present study, we aimed to analyse the biological function of the pSer68-PLM-NCX1 interaction by developing high-affinity blocking peptides. PLM was observed to co-fractionate and co-immunoprecipitate with NCX1 in rat left ventricle, and in co-transfected HEK (human embryonic kidney)-293 cells. For the first time, the NCX1-PLM interaction was also demonstrated in the brain. PLM binding sites on NCX1 were mapped to two regions by peptide array assays, containing the previously reported PASKT and QKHPD motifs. Conversely, the two NCX1 regions bound identical sequences in the cytoplasmic domain of PLM, suggesting that NCX1-PASKT and NCX1-QKHPD might bind to each PLM monomer. Using two-dimensional peptide arrays of the native NCX1 sequence KHPDKEIEQLIELANYQVLS revealed that double substitution of tyrosine for positions 1 and 4 (K1Y and D4Y) enhanced pSer68- PLM binding 8-fold. The optimized peptide blocked binding of NCX1-PASKT and NCX1-QKHPD to PLM and reversed PLM(S68D) inhibition of NCX1 activity (both forward and reverse mode) in HEK-293 cells. Altogether our data indicate that PLM interacts directly with NCX1 and inhibits NCX1 activity when phosphorylated at Ser68. [ABSTRACT FROM AUTHOR]

Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions, NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between the plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K+ in the regulation of NKA in skeletal muscle are highlighted. [ABSTRACT FROM AUTHOR]

FXYD proteins regulate Na + /K + -ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka ( Oryzias dancena ) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka -α was elevated 48 h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12 h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka -α mRNA expression and NKA activity increased significantly 12 h and 1 h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein–protein interaction between branchial NKA and FXYD11 increased acutely 12 h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1 h following post-exposure to SW, without the elevation of protein–protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts. [ABSTRACT FROM AUTHOR]

The present study examined the effect of intensive training in combination with marked reduction in training volume on phospholemman (FXYD1) expression and phosphorylation at rest and during exercise. Eight well-trained cyclists replaced their regular training with speed-endurance training (10 -12 × ~30-s sprints) two or three times per week and aerobic high-intensity training (4-5 × 3-4 min at 90-95% of peak aerobic power output) 1-2 times per week for 7 wk and reduced the training volume by 70%. Muscle biopsies were obtained before and during a repeated high-intensity exercise protocol, and protein expression and phosphorylation were determined by Western blot analysis. Expression of FXYD1 (30%), actin (40%), mammalian target of rapamycin (mTOR) (12%), phospholamban (PLN) (16%), and Ca2+calmodulin-dependent protein kinase II (CaMKII) (25%) was higher (P < 0.05) than before the training intervention. In addition, after the intervention, nonspecific FXYD1 phosphorylation was higher (P < 0.05) at rest and during exercise, mainly achieved by an increased FXYD1 Ser-68 phosphorylation, compared with before the intervention. CaMKII, Thr-287, and eukaryotic elongation factor 2 Thr-56 phosphorylation at rest and during exercise, overall PKCα/β , Thr-638/641, and mTOR Ser-2448 phosphorylation during repeated intense exercise as well as resting PLN Thr-17 phosphorylation were also higher (P < 0.05) compared with before the intervention period. Thus, a period of high-intensity training with reduced training volume increases expression and phosphorylation levels of FXYD1, which may affect Na+/K+ pump activity and muscle K+ homeostasis during intense exercise. Furthermore, higher expression of CaMKII and PLN, as well as increased phosphorylation of CaMKII Thr-287 may have improved intracellular Ca2+ handling. [ABSTRACT FROM AUTHOR]

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na + –K + –ATPase and L-type Ca 2 + channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca 2 + channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca 2 + ] i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca 2 + current (I Ca ) and sarcoplasmic reticulum (SR) Ca 2 + content but not Na + /Ca 2 + exchange current (I NaCa ) or SR Ca 2 + uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored I Ca amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD 50 and APD 90 were prolonged in shBAG3 myocytes. Protein levels of Ca 2 + entry molecules and other important excitation–contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca 2 + channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. [ABSTRACT FROM AUTHOR]

The sodium (Na+)-calcium (Ca2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca2+ homeostasis. Serine 68 phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na+/K+ ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1, however, the molecular basis of this association is unknown. In the present study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pull-down experiments and peptide arrays constrained PP1c anchoring to the K-I/V-F-F motif in the first Ca2+ binding domain (CBD1) in NCX1. This binding site is also partially in agreement with the extended PP1 binding motif; K-I/V-F-F-X5-8-Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K-I/V-F-F motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K-I/V-F-F motif was mutated or when the PLM and PP1c binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1-(F407P) (mutated K-I/V-F-F motif) resulted in NCX1 current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation. [ABSTRACT FROM AUTHOR]

The Na+-K+-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na+-K+-ATPase, plays a role in the acute stimulation of α2in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb+ transport by the α2-Na+-K+-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na+ affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation. [ABSTRACT FROM AUTHOR]

Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1β1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between theαNandαP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na+ -selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies. [ABSTRACT FROM AUTHOR]

We evaluated whether phospholemman (PLM) regulates L-type Ca 2 + current (I Ca ) in mouse ventricular myocytes. Expression of α 1 -subunit of L-type Ca 2 + channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak I Ca (at − 10 mV) from KO myocytes was ~ 41% larger, the inactivation time constant (τ inact ) of I Ca was ~ 39% longer, but deactivation time constant (τ deact ) was similar. In the presence of isoproterenol (1 μM), peak I Ca was ~ 48% larger and τ inact was ~ 144% higher in KO myocytes. With Ba 2 + as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τ deact . To dissect the molecular determinants by which PLM regulated I Ca , we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24 h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak I Ca and longer τ inact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak I Ca . Expressing PLM mutants did not alter expression of α 1 -subunit of L-type Ca 2 + channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak I Ca amplitude. We conclude that PLM limits Ca 2 + influx in cardiac myocytes by reducing maximal I Ca and accelerating voltage-dependent inactivation. [ABSTRACT FROM AUTHOR]

Background: Phospholemman ( PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels ( LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods: The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type ( WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263 AA, 6869 AA, AAAA, 6263 DD, 6869 DD or DDDD. Results: WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation ( VDI). PLM mutants 6869 DD and DDDD significantly increased the peak of the currents. 6263 DD accelerated channel activation, while 6263 AA slowed it more than WT PLM. 6869 DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263 AA, and DDDD accelerated the channel VDI more than 6869 DD. Conclusions: Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects. [ABSTRACT FROM AUTHOR]

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump. [ABSTRACT FROM AUTHOR]

Aims Elevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force–frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM3SA knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression. Methods and results In wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM3SA mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na+]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM3SA myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM3SA mice compared with banded WT. Conclusions Decreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure. [ABSTRACT FROM PUBLISHER]

By interacting with Na+, K+-ATPase (NKA), the FXYD domain-containing ion transport regulator (FXYD) is involved in teleost osmoregulation, but knowledge of FXYD in marine fish is limited. In the present study, fxyd11 and fxyd12 were identified from the spotted scat (Scatophagus argus), and the two members of the FXYD protein family were expressed in a tissue-specific manner. Fxyd11 mRNA was predominantly expressed in gills, whereas fxyd12 mRNA was mainly distributed in kidneys and intestines. Acute hyposaline stress altered the activity of NKA and the expression of fxyd11 and fxyd12 in gills, kidneys, and intestines. Branchial fxyd11 mRNA expression remained at a low level during freshwater acclimation, whereas NKA activity increased, showing a negative correlation that differed from previous reports. Similarly, renal expression of fxyd11 and fxyd12 mRNA was negatively correlated with NKA activity. Unlike in gills and kidneys, intestinal NKA activity and mRNA expression of fxyd11 and fxyd12 were comparably suppressed. Taken together, the salinity-dependent expression of fxyd11 and fxyd12, and correlation with NKA activity suggested that both fxyd11 and fxyd12 were involved in the response to acute hyposaline challenge in the spotted scat. [ABSTRACT FROM AUTHOR]

Phospholemman (PLM), when phosphorylated at Ser68, 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 [Ca2+]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 ischemiareperfusion, 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. [ABSTRACT FROM AUTHOR]

Members of the FXYD domain-containing ion transport regulator protein family, including FXYD3 and FXYD5, play an important role in the pathogenesis of numerous tumors. However, the correlation between the expression of FXYD6 and tumors remains poorly understood. In the current study, the expression of FXYD6 was examined immunohistochemically in 72 cholangiocarcinoma tissues and 30 distal normal bile duct tissues matched with the tumors. The results show that the positive expression rate of FXYD6 was significantly higher in cholangiocarcinoma than that in normal bile duct tissue (69 vs. 33.3%; P=0.002). Furthermore, the positive expression rate of FXYD6 in well- and moderately-differentiated cholangiocarcinoma was clearly higher than that in poorly-differentiated and mucinous cholangiocarcinoma (85.7 vs. 40%; P=0.000). However, there was no significant correlation between the expression of FXYD6 and gender (P=0.393), age (P=0.174), histological type (P=0.123), T stage (P=0.164), lymph node metastasis (P=0.343), perineural invasion (P=0.088) and tumor location (P=0.238). The results of this study indicate that FXYD6 may be a new biomarker for cholangiocarcinoma and may be associated with a favorable prognosis in this malignant disease. [ABSTRACT FROM AUTHOR]

The human α 1/His 10-β 1 isoform of Na,K-ATPase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na + concentration ( K1/2) of Na + ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the negatively charged lipid DOPS (<10 %), FXYD1 increases K1/2 of Na + ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concentrations of DOPS (>10 %), little or no effect of FXYD1 on the K1/2 of Na + ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K1/2 of Na + ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the α-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane. [ABSTRACT FROM AUTHOR]

Highlights: [•] Inhibition of Na+/K+ ATPase activity upon TxA2 mimetic U46619 stimulation. [•] Involvement of 70kDa inhibitor protein under U46619 mediated oxidative stress. [•] Isoform specific inhibition Na+/K+ ATPase by oxidants. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (iCell Cardiomyocytes) with ion channel activities that are remarkably similar to adult cardiomyocytes. Here, we extend this characterization to cardiac ion transporters. Additionally, we document facile molecular biological manipulation of iCell Cardiomyocytes to overexpress and knockdown transporters and regulatory proteins. Na/Ca exchange (NCX1) and Na/K pump currents were recorded via patch clamp, and Na/H and Cl/OH exchanges were recorded via oscillating proton-selective microelectrodes during patch clamp. Flux densities of all transport systems are similar to those of nonrodent adult cardiomyocytes. NCX1 protein and NCX1 currents decline after NCX1 small interfering (si)RNA transfection with similar time courses (τ ≈ 2 days), and an NCX1-Halo fusion protein is internalized after its extracellular labeling by AlexaFluor488 Ligand with a similar time course. Loss of the cardiac regulatory protein phospholemman (PLM) occurs over a longer time course (τ ≈ 60 h) after PLM small interfering RNA transfection. Similar to multiple previous reports for adult cardiomyocytes, Na/K pump currents in iCell Cardiomyocytes are not enhanced by activating cAMP production with either maximal or submaximal cytoplasmic Na and using either forskolin or isoproterenol to activate adenylate cyclases. Finally, we describe Ca influx-dependent changes of iCell Cardiomyocyte capacitance (Cm). Large increases of Cm occur during Ca influx via NCX1, thereby documenting large internal membrane reserves that can fuse to the sarcolemma, and subsequent declines of Cm document active endocytic processes. Together, these results document a great potential of iCell Cardiomyocytes for both short- and long-term studies of cardiac ion transporters and their regulation. [ABSTRACT FROM AUTHOR]