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Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson's disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H+,K+-ATPase. The K+-dependent ATPase activity and the lysosomal K+-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, thapsigargin, and K+-competitive inhibitors of gastric H+,K+-ATPase, such as vonoprazan and SCH28080. Interestingly, these H+,K+-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H+/K+-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes. Mutations in the human ATP13A2, a lysosomal ATPase, is associated with pathogenesis of Parkinson's disease. Here, the authors show that ATP13A2 functions as H + /K + transporting protein, preventing lysosomal alkalinization and α-synuclein accumulation. [ABSTRACT FROM AUTHOR]

The gastric proton pump (H + ,K + -ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH 5). Interestingly, the enzymatic activity of H + ,K + -ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H + ,K + -ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H 2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H + ,K + -ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK. [ABSTRACT FROM AUTHOR]

Highlights: [•] ERp57 is abundantly expressed in the apical membrane of gastric parietal cells. [•] ERp57 positively regulates H+,K+-ATPase activity apart from its chaperoning function. [•] ERp57 has no effect on Na+,K+-ATPase activity. [ABSTRACT FROM AUTHOR]

Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl- cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase 1-subunit (α1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous α1NaK, and it recruited α1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of α1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with α1NaK. In conclusion, KCC3a forms a functional complex with α1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates α1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells. [ABSTRACT FROM AUTHOR]

Gastric H+,K+ATPase is shown to transport 2 mol of H+/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H+ transport mechanism is due to charge transfer and/or transfer of hydronium ion (H3O+). From transport of [18O] H2O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H+,K+-ATPase α-subunit at E1 conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H3O+- Lys-164-Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H3O+-Gln-161-Gln-345) in mutant K164L and (H3O+-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H+,K+-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H3O+-Asn-794-Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H2O in sites 2 and 1, the H2O comes from cytosolic medium, and H3O+ in the sites are transported into lumen during the conformational transition from E1P to E2P. [ABSTRACT FROM AUTHOR]

To investigate the inhibitory effect of a commercial proton pump inhibitor (lansoprazole) on the gastric proton pump H + ,K + -ATPase in vitro, we used orally disintegrating (OD) tablets including original brand-name and generic tablets. In the course of the development of generic products, dissolution and clinical tests are necessary to ensure their bioequivalence to the original brand-name products; by contrast, there is almost no opportunity to demonstrate their activity in vitro. This study initially compared the similarity of the dissolution of test generic tablets with that of the original brand-name tablets. The dissolution tests for 15 and 30-mg lansoprazole tablets found their dissolution properties were similar. Subsequently, the dissolution media were sampled and then their effects on the H + ,K + -ATPase activity were measured using tubulovesicles prepared from the gastric mucosa of hogs. We confirmed that the inhibitory effects of the generic tablets on H + ,K + -ATPase activity were consistent with those of the original brand-name tablets. Furthermore, lansoprazole contents in each tablet estimated from their inhibitory effects were in good agreement with their active pharmaceutical ingredient content. To our knowledge, this is the first technical report to compare the in vitro biochemical activity of lansoprazole OD tablets between the original brand-name and generic commercial products.

It has been reported that chloride-proton exchanger ClC-5 and vacuolar-type H(+)-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H(+),K(+)-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of (36)Cl(-) into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H(+),K(+)-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H(+),K(+)-ATPase, ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase, but not with endogenous Na(+),K(+)-ATPase. The SCH28080-sensitive (36)Cl(-) transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H(+) transport activity, did not show the SCH28080-sensitive (36)Cl(-) transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H(+),K(+)-ATPase. Our results suggest that ClC-5 and H(+),K(+)-ATPase are functionally associated and that they may contribute to gastric acid secretion.

TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl(-) channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca(2+) ionophore, ionomycin, or by an increase in the intracellular free Ca(2+) concentration. The free Ca(2+) concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca(2+) level, in contrast to TMEM16A/B currents. The Ca(2+)-activated TMEM16F currents were anion selective, because replacing Cl(-) with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I(-) > Br(-) > Cl(-) > F(-) > aspartate(-). Niflumic acid, a Ca(2+)-activated Cl(-) channel blocker, inhibited the TMEM16F-dependent Cl(-) currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl(-) channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca(2+)-activated Cl(-) channel with a Ca(2+) sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.