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.

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.

Localization of Na(+)/K(+)-ATPase in silkworm brain: a possible mechanism for protection of Na(+)/K(+)-ATPase from Ca(2+).

In mammalian blood, the Na(+) concentration is higher than the K(+) concentration, whereas in hemolymph of lepidopterous insects, the K(+) concentration is higher than the Na(+) concentration. Na(+)/K(+)-ATPase regulates Na(+) and K(+) concentrations in mammalian blood. Therefore, the absence of Na(+)/K(+)-ATPase in lepidopterous insects might be expected. However, we have observed that Na(+)/K(+)-ATPase is abundant in nerve tissues of larvae of silkworm, a lepidopterous insect. Furthermore, we found that silkworm Na(+)/K(+)-ATPase was completely inhibited by 3 mM Ca(2+)in vitro (Homareda, 2010), although the Ca(2+) concentration is very high (30-50 mM) in the hemolymph of silkworm larvae. To investigate the reason why silkworm Na(+)/K(+)-ATPase is not inhibited by Ca(2+)in vivo, we observed the localization of Na(+)/K(+)-ATPase in nerve tissues using immunohistochemical techniques. Na(+)/K(+)-ATPase was distributed in the cortex and neuropile but not in the perineurium of the silkworm brain, while plasma membrane Ca(2+)-ATPase appeared to distribute in the perineurium as well as in the cortex and neuropile. These results support a possibility that neuronal Na(+)/K(+)-ATPase is protected from a high Ca(2+) concentration by the blood-brain barrier consisting of perineurial glial cells with plasma membrane Ca(2+)-ATPase.

Properties of silkworm Na+/K+-ATPase.

The high Na(+) and low K(+) concentrations in mammalian blood are maintained by Na(+)/K(+)-ATPase. In contrast, the K(+) concentration is higher than the Na(+) concentration in the hemolymph of the silkworm Bombyx mori, a Lepidopterous insect. Although Na(+)/K(+)-ATPase, therefore, appears not to be in silkworm, we confirmed the presence of Na(+)/K(+)-ATPase in nerve tissues of silkworm but not in skeletal muscle or the dorsal vessel. The enzymatic properties of silkworm Na(+)/K(+)-ATPase were characterized in detail and compared with those of dog Na(+)/K(+)-ATPase. Silkworm Na(+)/K(+)-ATPase had a much lower affinity for K(+) and a somewhat higher affinity for Na(+) than dog Na(+)/K(+)-ATPase. The optimal temperature of silkworm Na(+)/K(+)-ATPase activity was lower than that of dog Na(+)/K(+)-ATPase. The optimal Mg(2+) concentration, pH and sensitivities to Ca(2+) and ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, of the two ATPases were identical. These results indicate that the enzymatic properties of the silkworm Na(+)/K(+)-ATPase are suitable for its growth, despite the differences between dog and silkworm Na(+)/K(+)-ATPases. Antisera raised against dog Na(+)/K(+)-ATPase recognized only the α-subunit of silkworm Na(+)/K(+)-ATPase.

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.