Regulation of the Cardiac Na+/K+ ATPase by Phospholemman

Hansraj Dhayan, Rajender Kumar, Andreas Kukol, ‘Regulation of the Cardiac Na+/K+ ATPase by Phospholemman’, in Sajal Chakraborti, Naranjan Dhalla, eds., Regulation of Membrane Na+-K+ ATPase, (Switzerland: Springer, 2016), ISBN 978-3-319-24748-9, eISBN 978-3-319-24750-2.Peer reviewe

Expression Profile of Nuclear Receptors along Male Mouse Nephron Segments Reveals a Link between ERRβ and Thick Ascending Limb Function

The nuclear receptor family orchestrates many functions related to reproduction, development, metabolism, and adaptation to the circadian cycle. The majority of these receptors are expressed in the kidney, but their exact quantitative localization in this ultrastructured organ remains poorly described, making it difficult to elucidate the renal function of these receptors. In this report, using quantitative PCR on microdissected mouse renal tubules, we established a detailed quantitative expression map of nuclear receptors along the nephron. This map can serve to identify nuclear receptors with specific localization. Thus, we unexpectedly found that the estrogen-related receptor β (ERRβ) is expressed predominantly in the thick ascending limb (TAL) and, to a much lesser extent, in the distal convoluted tubules. In vivo treatment with an ERR inverse agonist (diethylstilbestrol) showed a link between this receptor family and the expression of the Na+,K+-2Cl− cotransporter type 2 (NKCC2), and resulted in phenotype presenting some similarities with the Bartter syndrom (hypokalemia, urinary Na+ loss and volume contraction). Conversely, stimulation of ERRβ with a selective agonist (GSK4716) in a TAL cell line stimulated NKCC2 expression. All together, these results provide broad information regarding the renal expression of all members of the nuclear receptor family and have allowed us to identify a new regulator of ion transport in the TAL segments

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Properties and expression of Na+/K+-ATPase α-subunit isoforms in the brain of the swamp eel, Monopterus albus, which has unusually high brain ammonia tolerance

10.1371/journal.pone.0084298PLoS ONE812-POLN

Hypertonicity counteracts MCL 1 and renders BCL XL a synthetic lethal target in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and difficult‐to‐treat cancer entity. Current therapies ultimately aim to activate the mitochondria‐controlled (intrinsic) apoptosis pathway, but complex alterations in intracellular signaling cascades and the extracellular microenvironment hamper treatment response. On the one hand, proteins of the BCL‐2 family set the threshold for cell death induction and prevent accidental cellular suicide. On the other hand, controlling a cell's readiness to die also determines whether malignant cells are sensitive or resistant to anticancer treatments. Here, we show that HNSCC cells upregulate the proapoptotic BH3‐only protein NOXA in response to hyperosmotic stress. Induction of NOXA is sufficient to counteract the antiapoptotic properties of MCL‐1 and switches HNSCC cells from dual BCL‐XL/MCL‐1 protection to exclusive BCL‐XL addiction. Hypertonicity‐induced functional loss of MCL‐1 renders BCL‐XL a synthetically lethal target in HNSCC, and inhibition of BCL‐XL efficiently kills HNSCC cells that poorly respond to conventional therapies. We identify hypertonicity‐induced upregulation of NOXA as link between osmotic pressure in the tumor environment and mitochondrial priming, which could perspectively be exploited to boost efficacy of anticancer drugs

New molecular determinants controlling the accessibility of ouabain to its binding site in human Na,K-ATPase alpha isoforms

Inhibition of Na,K-ATPase alpha2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of alpha1 isoforms may be responsible for their toxic effects. Human Na,K-ATPase alpha1 and alpha2 isoforms exhibit a high ouabain affinity but significantly differ in the ouabain association and dissociation rates. To identify the structural determinants that are involved in these differences, we have prepared chimeras between human alpha1 and alpha2 isoforms and alpha2 mutants in which nonconserved amino acids were exchanged with those of the alpha1 isoform, expressed these constructs in Xenopus laevis oocytes, and measured their ouabain binding kinetics. Our results show that replacement of Met119 and Ser124 in the M1-M2 extracellular loop of the alpha2 isoform by the corresponding Thr119 and Gln124 of the alpha1 isoform shifts both the fast ouabain association and dissociation rates of the alpha2 isoform to the slow ouabain binding kinetics of the alpha1 isoform. The amino acids at position 119 and 124 cooperate with the M7-M8 hairpin and are also responsible for the small differences in the ouabain affinity of the ouabain-sensitive alpha1 and alpha2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase alpha-subunit that are involved in ouabain binding and probably control, in an alpha isoform-specific way, the access and release of ouabain to and from its binding site

Electrogenicity of Na,K- and H,K-ATPase activity and presence of a positively charged amino acid in the fifth transmembrane segment.

The transport activity of the Na,K-ATPase (a 3 Na+ for 2 K+ ion exchange) is electrogenic, whereas the closely related gastric and non-gastric H,K-ATPases perform electroneutral cation exchange. We have studied the role of a highly conserved serine residue in the fifth transmembrane segment of the Na,K-ATPase, which is replaced with a lysine in all known H,K-ATPases. Ouabain-sensitive 86Rb uptake and K+-activated currents were measured in Xenopus oocytes expressing the Bufo bladder H,K-ATPase or the Bufo Na,K-ATPase in which these residues, Lys800 and Ser782, respectively, were mutated. Mutants K800A and K800E of the H,K-ATPase showed K+-stimulated and ouabain-sensitive electrogenic transport. In contrast, when the positive charge was conserved (K800R), no K+-induced outward current could be measured, even though rubidium transport activity was present. Conversely, the S782R mutant of the Na,K-ATPase had non-electrogenic transport activity, whereas the S782A mutant was electrogenic. The K800S mutant of the H,K-ATPase had a more complex behavior, with electrogenic transport only in the absence of extracellular Na+. Thus, a single positively charged residue in the fifth transmembrane segment of the alpha-subunit can determine the electrogenicity and therefore the stoichiometry of cation transport by these ATPases

Bufo marinus bladder H-K-ATPase carries out electroneutral ion transport

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K(+) into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K(+) activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of approximately 20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO at a PCO(2) of 40 Torr, the negative current activated by K(+) was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport