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P-type ion pumps (P-type ATPases) are involved in various fundamental biological processes. For example, the gastric proton pump (H + ,K + -ATPase) and sodium pump (Na + ,K + -ATPase) are responsible for secretion of gastric acid and maintenance of cell membrane potential, respectively. In this review, we summarize three topics of our studies. The first topic is gastric H + ,K + -ATPase associated with Cl - -transporting proteins (Cl - /H + exchanger ClC-5 and K + -Cl - cotransporter KCC4). In gastric parietal cells, we found that ClC-5 is predominantly expressed in intracellular tubulovesicles and that KCC4 is predominantly expressed in the apical membrane. Gastric acid (HCl) secretion may be accomplished by the two different complexes of H + ,K + -ATPase and Cl - -transporting protein. The second topic focuses on the Na + ,K + -ATPase α1-isoform (α1NaK) associated with the volume-regulated anion channel (VRAC). In the cholesterol-enriched membrane microdomains of human cancer cells, we found that α1NaK has a receptor-like (non-pumping) function and that binding of low concentrations (nM level) of cardiac glycosides to α1NaK activates VRAC and exerts anti-cancer effects without affecting the pumping function of α1NaK. The third topic is the Na + ,K + -ATPase α3-isoform (α3NaK) in human cancer cells. We found that α3NaK is abnormally expressed in the intracellular vesicles of attached cancer cells and that the plasma membrane translocation of α3NaK upon cell detachment contributes to the survival of metastatic cancer cells. Our results indicate that multiple functions of P-type ion pumps are generated by different membrane environments and their associated proteins.

Proton(H+) of gastric acid(HCl) is actively secreted by gastric proton pump (H+, K+- ATPase) in the parietal cells. The proton pump is expressed in both tubulovesicles and apical membrane of the cells. In resting parietal cells, tubulovesicles are present in intracellular compartments underlying the apical membrane and forming a reticulated meshwork. Upon stimulation, tubulovesicles fuse each other and connect with the apical membrane, resulting in massive acid secretion. On the other hand, the mechanism of apical Cl- transport for HCl secretion is not fully understood, although several Cl- transporters and Cl- channels have been reported to be the candidate. Here, we summarized the function of Cl- transporters such as KCC4, a K+-Cl- cotransporter, and ClC-5, a Cl-/H+ exchanger, in gastric acid secretion.

Gastric proton pump (H⁺, K⁺-ATPase) secretes H⁺ of acid (HCl) via the luminal membrane of parietal cells. For the HCl secretion, Cl⁻- and K⁺-transporting proteins are required. Recent our studies have demonstrated that K⁺-Cl⁻ cotransporters (KCC3a and KCC4) are expressed in gastric parietal cells. KCC3a is associated with Na⁺, K⁺-ATPase in the basolateral membrane, and KCC4 is associated with H⁺, K⁺-ATPase in the apical canalicular membrane. This paper summarizes the functional association between KCCs and P-type ATPases and the contribution of these complexes to acid secretion in gastric parietal cells.

The gastric proton pump (H+,K+-ATPase) responsible for the H+ secretion of gastric acid is an essential therapeutic target for acid-related diseases. H+,K+-ATPase belongs to a P 2 -type ATPase family. Here, we examined the effects of a newly synthesized dihydropyrazole derivative KYY-008 on the H+,K+-ATPase. KYY-008 concentration-dependently inhibited the enzyme activity of the ATPase in the membrane fractions prepared from isolated hog gastric mucosa and from human kidney HEK293 cells in which gastric H+,K+-ATPase is exogenously expressed. The IC 50 values in these samples were 3.4 μM and 3.7 μM, respectively. In addition, KYY-008 significantly inhibited the H+,K+-ATPase-derived H+ uptake into the tightly sealed vesicles prepared from the hog gastric mucosa. In contrast, KYY-008 has no effect on the activities of other P 2 -type ATPases such as Na+,K+-ATPase and Ca2+-ATPase. KYY-008 did not change the ionic currents of voltage-dependent Ca2+ channels, that were potential targets for some dihydropyrazole derivatives. Together, we discovered a new dihydropyrazole derivative which acts as a selective inhibitor of gastric H+,K+-ATPase. • New dihydropyrazole derivative KYY-008 inhibited enzyme activity of gastric H+,K+-ATPase. • KYY-008 suppressed the H+,K+-ATPase-derived H+ transport in gastric vesicles. • KYY-008 had no effect on Na+,K+-ATPase, Ca2+-ATPase, and voltage-dependent Ca2+ channels. [ABSTRACT FROM AUTHOR]

In the stomach, Sonic Hedgehog (Shh) is highly expressed in gastric parietal cells, and acts as a morphogen in early development of the organ. Here, we found that the cleaved N-terminal fragment of Shh (Shh-N) was abundantly expressed in hog gastric vesicles derived from the apical membrane of parietal cells. Interestingly, Shh-N recombinant significantly decreased K+-dependent ATP-hydrolyzing activity, which is sensitive to an inhibitor of H+,K+-ATPase (SCH28080), in hog gastric tubulovesicles and membrane fractions of the H+,K+-ATPase-expressing cells. In the living cells, Shh-N recombinant inhibited the SCH28080-sensitive 86Rb+-uptake. Together, Shh-N may directly bind to extracellular side of H+,K+-ATPase, and negatively regulates the pump activity. This is the first report to explore non-morphogenic property of Shh on ion transporters. • Sonic Hedgehog (Shh), a morphogen, was cleaved in apical membrane vesicles rich in gastric H+,K+-ATPase. • The cleaved N-terminal fragment of Shh (Shh-N) inhibited the H+,K+-ATPase activity. • Extracellular Shh-N inhibited the K+-transport activity of H+,K+-ATPase in living cells. • Shh-N was found to act as a non-morphogenic inhibitor of gastric H+,K+-ATPase. [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]