ß subunit affects Na+ and K+ affinities of Na+/K+-ATPase: Na+ and K+ affinities of a hybrid Na+/K+-ATPase composed of insect α and mammalian ß subunits.

The affinity for K+ of silkworm Na+/K+-ATPase, which is composed of α and ß subunits, is remarkably lower than that of mammalian Na+/K+-ATPase, with a slightly higher affinity for Na+. Because the α subunit had more than 70% identity to the mammalian α subunit in the amino acid sequence, whereas the ß subunit, a glycosylated protein, had less than 30% identity to the mammalian ß subunit, it was suggested that the ß subunit was involved in the affinities for Na+ and K+ of Na+/K+-ATPase. To confirm this hypothesis, we examined whether replacing the silkworm ß subunit with the mammalian ß subunit affected the affinities for Na+ and K+ of Na+/K+-ATPase. Cloned silkworm α and cloned rat ß1 were co-expressed in BM-N cells, a cultured silkworm ovary-derived cell lacking endogenous Na+/K+-ATPase, to construct a hybrid Na+/K+-ATPase, in which the silkworm ß subunit was replaced with the rat ß1 subunit. The hybrid Na+/K+-ATPase increased the affinity for K+ by 4.1-fold and for Na+ by 0.65-fold compared to the wild-type one. Deglycosylation of the silkworm ß subunit did not affect the K+ affinity. These results support the involvement of the ß subunit in the Na+ and K+ affinities of Na+/K+-ATPase.

Golgi stress induces upregulation of the ER-Golgi SNARE Syntaxin-5, altered ßAPP processing, and Caspase-3-dependent apoptosis in NG108-15 cells.

The involvement of secretory pathways and Golgi dysfunction in neuronal cells during Alzheimer's disease progression is poorly understood. Our previous overexpression and knockdown studies revealed that the intracellular protein level of Syntaxin-5, an endoplasmic reticulum-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE), modulates beta-amyloid precursor protein processing in neuronal cells. We recently showed that changes in endogenous Syntaxin-5 protein expression occur under stress induction. Syntaxin-5 was upregulated by endoplasmic reticulum stress but was degraded by Caspase-3 during apoptosis in neuronal cells. In addition, we showed that sustained endoplasmic reticulum stress promotes Caspase-3-dependent apoptosis during the later phase of the endoplasmic reticulum stress response in NG108-15 cells. In this study, to elucidate the consequences of secretory pathway dysfunction in beta-amyloid precursor protein processing that lead to neuronal cell death, we examined the effect of various stresses on endoplasmic reticulum-Golgi SNARE expression and beta-amyloid precursor protein processing. By using compounds to disrupt Golgi function, we show that Golgi stress promotes upregulation of the endoplasmic reticulum-Golgi SNARE Syntaxin-5, and prolonged stress causes Caspase-3-dependent apoptosis. Golgi stress induced intracellular beta-amyloid precursor protein accumulation and a concomitant decrease in total amyloid-beta production. We also examined the protective effect of the chemical chaperone 4-phenylbutylate on changes in amyloid-beta production and the activation of Caspase-3 induced by endoplasmic reticulum and Golgi stress. The compound alleviated the increase in the amyloid-beta 1-42/amyloid-beta 1-40 ratio induced by endoplasmic reticulum and Golgi stress. Furthermore, 4-phenylbutylate could rescue Caspase-3-dependent apoptosis induced by prolonged organelle stress. These results suggest that organelle stress originating from the endoplasmic reticulum and Golgi has a substantial impact on the amyloidogenic processing of beta-amyloid precursor protein and Caspase-3-dependent apoptosis, leading to neuronal cell death.

Inhibition of the human secretory pathway Ca2+, Mn2+-ATPase1a by 1,3-thiazole derivatives.

The human Golgi/secretory pathway Ca2+,Mn2+-ATPase 1 (hSPCA1) transports Ca2+ and Mn2+ into the Golgi lumen. Studies of the biological functions of hSPCA1 are limited by a lack of selective pharmacological tools for SPCA1 inhibition. The aim of this study was therefore to identify compounds that specifically inhibit hSPCA1 activity. We found that five 1,3-thiazole derivatives exhibited inhibitory action towards the ATP hydrolysis activity of hSPCA1a in a concentration-dependent manner. Among the derivatives tested, compound 1 was the most potent, completely inhibiting hSPCA1a activity with a half-maximal inhibition (IC50) value of 0.8 µM. Compound 1 also partially inhibited the activity of another Ca2+,Mn2+-ATPase (hSPCA2) and Ca2+-ATPase (rSERCA1a), but had no effect on Na+,K+-ATPase or H+,K+-ATPase. Treatment of HeLa cells with compound 1 led to fragmentation of the Golgi ribbon into smaller stacks. In addition, compound 1 mobilized intracellular Ca2+ in HeLa cells that had been pre-treated with thapsigargin. Therefore, based on its selectivity and potency, compound 1 may be a valuable tool with which to further explore the role of SPCA1 in cellular processes.

A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and ß subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and ß subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the ß subunit with mammalian counterparts was as low as 30%. Cloned α and ß cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the ß subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

Inhibitory action of linoleamide and oleamide toward sarco/endoplasmic reticulum Ca2+-ATPase.

BACKGROUND: SERCA maintains intracellular Ca2+ homeostasis by sequestering cytosolic Ca2+ into SR/ER stores. Two primary fatty acid amides (PFAAs), oleamide (18:19-cis) and linoleamide (18:29,12-cis), induce an increase in intracellular Ca2+ levels, which might be caused by their inhibition of SERCA. METHODS: Three major SERCA isoforms, rSERCA1a, hSERCA2b, and hSERCA3a, were individually overexpressed in COS-1 cells, and the inhibitory action of PFAAs on Ca2+-ATPase activity of SERCA was examined. RESULTS: The Ca2+-ATPase activity of each SERCA was inhibited in a concentration-dependent manner strongly by linoleamide (IC50 15-53µM) and partially by oleamide (IC50 8.3-34µM). Inhibition by other PFAAs, such as stearamide (18:0) and elaidamide (18:19-trans), was hardly or slightly observed. With increasing dose, linoleamide decreased the apparent affinity for Ca2+ and the apparent maximum velocity of Ca2+-ATPase activity of all SERCAs tested. Oleamide also lowered these values for hSERCA3a. Meanwhile, oleamide uniquely reduced the apparent Ca2+ affinity of rSERCA1a and hSERCA2b: the reduction was considerably attenuated above certain concentrations of oleamide. The dissociation constants for SERCA interaction varied from 6 to 45µM in linoleamide and from 1.6 to 55µM in oleamide depending on the isoform. CONCLUSIONS: Linoleamide and oleamide inhibit SERCA activity in the micromolar concentration range, and in a different manner. Both amides mainly suppress SERCA activity by lowering the Ca2+ affinity of the enzyme. GENERAL SIGNIFICANCE: Our findings imply a novel role of these PFAAs as modulators of intracellular Ca2+ homeostasis via regulation of SERCA activity.

Identification of novel inhibitors of human SPCA2.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 µM and 3.8 µM, respectively.

The involvement of L-type amino acid transporters in theanine transport.

L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms.

The dimeric form of Ca2+-ATPase is involved in Ca2+ transport in the sarcoplasmic reticulum.

To identify the functional unit of Ca(2+)-ATPase in the sarcoplasmic reticulum, we assessed Ca(2+)-transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca(2+)-ATPase molecules. We prepared heterodimers, consisting of a native Ca(2+)-ATPase molecule and a Ca(2+)-ATPase molecule inactivated by FITC labelling, by fusing vesicles loaded with each type of Ca(2+)-ATPase. The heterodimers exhibited neither Ca(2+) transport nor ATP hydrolysis, suggesting that Ca(2+) transport by the Ca(2+)-ATPase requires an interaction between functional Ca(2+)-ATPase monomers. This finding implies that the functional unit of the Ca(2+)-ATPase is a dimer.

Synthesis of ATP by the simple addition of ADP to the p-nitrophenyl phosphate-prepared phosphoenzyme of the sarcoplasmic reticulum Ca2+-ATPase.

The sarcoplasmic reticulum Ca2+-ATPase was phosphorylated with either p-nitrophenyl phosphate (pNPP) or with ATP in the presence of Ca2+, under the condition that the free energy change of pNPP hydrolysis is less than that of ATP, DeltaG'(pNPP)

Stimulation of p-nitrophenylphosphatase activity of Na+/K+-ATPase by NaCl with oligomycin or ATP.

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain-sensitive and K+-dependent p-nitrophenylphosphatase (pNPPase) activity of Na+/K+-ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+-binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+-ATPase by p-nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+-occluded E1 state of Na+/K+-ATPase, it seemed that the Na+-occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high-energy phosphoenzyme formed from ATP with Na+, is also the Na+-occluded E1 state, it is suggested that the Na+-occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between alpha subunits. Therefore, membrane-bound Na+/K+-ATPase would function as at least an (alphabeta)2-diprotomer with interacting alpha subunits at the phosphorylation step.

Ca(2+) transport of Ca(2+)-ATPase of the sarcoplasmic reticulum in an ADP-sensitive phosphoenzyme state.

To understand the energetics of Ca(2+)-transporting adenosine triphosphatase (Ca(2+)-ATPase), it is important to determine the energy consumption step. To do this, we measured the dissociation of Ca(2+) from Ca(2+)-ATPase into Ca(2+)-loaded vesicles. We observed that 45Ca(2+) added to the outside of the vesicles accumulated in the 40Ca(2+)-loaded vesicles after the addition of ADP and ATP. The acceleration of 45Ca(2+) accumulation increased by 1.4-fold after the addition of 1 microM ADP. Under the same conditions, Ca(2+)-dependent phosphate liberation was not observed, and all of the active phosphoenzymes were in the ADP-sensitive phosphoenzyme (E(1)P) state. These results indicated that the ADP stimulated 45Ca(2+) accumulation by the ADP-ATP exchange reaction and that this ADP-ATP exchange reaction did not pass through the ADP-insensitive phosphoenzyme state. Therefore, we demonstrate that one Ca(2+) ion dissociates at the E(1)P state, which does not correspond with the phosphoenzyme conversion, that is the energy consumption step in the E(1)-E(2) model.