On the reversibility of binding of cardiotonic steroids to a partially purified (Na + K)-activated adenosinetriphosphatase from beef brain

244

178

The vesicular gastric H,K-ATPase catalyzes an electroneutral H for K exchange allowing acidification of the intravesicular space. There is a total of 28 cysteines present in the ␣ subunit of the gastric H,K-ATPase, of which 10 are found in the predicted transmembrane segments and their connecting loop, and 9 are present in the ␤ subunit, of which 6 are disulfide-linked. To determine which of these was accessible to extracytoplasmic attack, the enzyme was inhibited by four different sub- , under acid transporting conditions. All of these compounds are weak bases that accumulate in the acidic space generated by the pump and undergo an acid catalyzed rearrangement to a cationic sulfenamide, which forms disulfides with accessible cysteines. The relative rates of acid activation of these compounds corresponded to the relative rates of inhibition of ATPase activity and acid transport. Fragmentation of the enzyme by trypsin followed by SDS-polyacrylamide gel electrophoresis showed that omeprazole bound covalently to one of the two cysteines in the domains containing the fifth and sixth transmembrane segments and their extracytoplasmic loop and to cysteine 892 in the loop between the seventh and eighth transmembrane segments, but inhibition correlated with the reaction with cysteines in the fifth and sixth domain. Lansoprazole bound to the cysteines in these two domains as well as to cysteine 321 toward the extracytoplasmic end of the third transmembrane segments. Pantoprazole bound only to either cysteine 813 or 822 in the fifth and sixth transmembrane region. The inhibition of Rabeprazole correlated also with its binding to this part of the protein, but this compound continued to bind after full inhibition, eventually binding also to cysteines 321 and 892. No binding was found to any of the cysteines in the seventh to tenth transmembrane segments. Thermolysin digestion of the isolated omeprazole-labeled fifth and sixth transmembrane pair showed that cysteine 813 was the site of labeling. It is concluded that binding of these sided reagents to cysteine 813 in the loop between transmembrane (TM)5 and TM6 is sufficient for inhibition of ATPase activity and acid transport by the gastric acid pump. Of the 10 cysteines present in the membrane and extracytoplasmic domain, only three are exposed sufficiently to allow reactivity with these cationic thiol reagents. The binding to cysteine 813 defines the location of the extracytoplasmic loop between TM5 and TM6 and places the carboxylic acids 820 and 824 conserved between the gastric H,K-and the Na,K-ATPases in TM6, consistent with their assumed role in cation binding.

Section: Methodsmentioning

confidence: 99%

Sites of Reaction of the Gastric H,K-ATPase with Extracytoplasmic Thiol Reagents

Besancon

Simon²,

Sachs

et al. 1997

244

178

“…After incubation at 37°C for 30 min, the inorganic phosphate released was measured as described elsewhere (29). The K ϩ -ATPase activity was calculated as the difference between activities in the presence and absence of KCl.…”

Section: Cavity Structure Of the Docking Site In Gastric Proton Pumpmentioning

confidence: 99%

The Cavity Structure for Docking the K+-competitive Inhibitors in the Gastric Proton Pump

Asano¹,

Yoshida

Yashiro

et al. 2004

2-Methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) is a reversible inhibitor specific for the gastric proton pump. The inhibition pattern is competitive with K ؉ . Here we studied the binding sites of this inhibitor on the putative three-dimensional structure of the gastric proton pump ␣-subunit that was constructed by homology modeling based on the structure of sarcoplasmic reticulum Ca 2؉ pump. Alanine and serine mutants of Tyr 801 located in the fifth transmembrane segment of the gastric proton pump ␣-subunit retained the 86 Rb transport and K ؉ -dependent ATPase (K ؉ -ATPase) activities. These mutants showed 60 -80-times lower sensitivity to SCH 28080 than the wild type in the 86 Rb transport activity. The K ؉ -ATPase activities of these mutants were not completely inhibited by SCH 28080. The sensitivity to SCH 28080 was dependent on the bulkiness of the side chain at this position. Therefore, the side chain of Tyr 801 is important for the interaction with this inhibitor. In the three-dimensional structure of the E 2 form (conformation with high affinity for K ؉ ) of the gastric proton pump, Tyr 801 faces a cavity surrounded by the first, fourth, fifth, sixth, and eighth transmembrane segments and fifth/sixth, seventh/eighth, and ninth/tenth loops. SCH 28080 can dock in this cavity. However, SCH 28080 cannot dock in the same location in the E 1 form (conformation with high affinity for proton) of the gastric proton pump due to the drastic rearrangement of the transmembrane helices between the E 1 and E 2 forms. These results support the idea that this cavity is the binding pocket of SCH 28080.The gastric H ϩ ,K ϩ -ATPase is a proton pump that is responsible for gastric acid secretion (1). This pump consists of two kinds of subunits, the ␣-and ␤-subunits. The ␣-subunit is the catalytic subunit containing 10 transmembrane segments. The ATP binding site (2, 3) and the cation binding sites are located on this subunit (4 -10). The ␤-subunit is the non-catalytic glycoprotein containing a single transmembrane segment and is involved in stabilization as well as targeting of the ␣-subunit to the plasma membrane (11). This pump belongs to the family of type II P-type ATPases, which include sarcoplasmic reticulum (SR) 1 Ca 2ϩ -ATPase (Ca 2ϩ pump) and Na ϩ ,K ϩ -ATPase (Na ϩ pump) (12).At present, inhibitors specific for the gastric proton pump are classified into two groups. Irreversible inhibitors such as omeprazole, rabeprazole, and lansoprazole, termed the proton pump inhibitors, are the first group. They are activated in acidic lumens, modify the cysteine residues located on the luminal side of the proton pump, and inhibit its pump activity. They are clinically used for controlling hyperacidity. The second classified group of specific proton pump inhibitors are the reversible inhibitors such as SCH 28080 (13) and 3-amino-5-methyl-2-(2-methyl-3-thienyl)imidazo[1,2-a]thieno[3,2-c]pyridine (SPI-447) (Fig. 1) (14), which are described as acid pump antagonists (APAs). They bind to the E 2 or E 2...

“…After incubation at 37°C for 30 min, the inorganic phosphate released was measured as described elsewhere (26). The K ϩ -ATPase activity was calculated as the difference between activities in the presence and absence of KCl.…”

mentioning

confidence: 99%

Alanine-scanning Mutagenesis of the Sixth Transmembrane Segment of Gastric H+,K+-ATPase α-Subunit

Asano¹,

Io²,

Kimura³

et al. 2001

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H ؉ ,K ؉ -ATPase ␣-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H ؉ ,K ؉ -ATPase; overall K ؉ -stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K ؉ -ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K ؉ , whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the ␣-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K ؉ -dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K ؉ -ATPase activity accompanying the decrease in the rate of phosphorylation.