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

Besancon, Marie; Simon, Alex; Sachs, George; Shin, Jai Moo

doi:10.1074/jbc.272.36.22438

Cited by 245 publications

(196 citation statements)

References 45 publications

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“…Although these effects could all be explained by ouabain binding elsewhere, it is intriguing that omeprazole inhibits gastric H͞K pumps, close relatives of Na͞K pumps, by reacting directly with the Cys analogous to the Na͞K pump's conserved TM6-Thr (42). And mutating that Thr to Cys renders the Na͞K pump susceptible to inhibition by omeprazole (31), while making it less sensitive to inhibition by ouabain (31,36).…”

Section: Discussionmentioning

confidence: 99%

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Artigas

Gadsby

2006

Proc. Natl. Acad. Sci. U.S.A.

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The Na͞K pump establishes essential ion concentration gradients across animal cell membranes. Cardiotonic steroids, such as ouabain, are specific inhibitors of the Na͞K pump. We exploited the marine toxin, palytoxin, to probe both the ion translocation pathway through the Na͞K pump and the site of its interaction with ouabain. Palytoxin uncouples the pump's gates, which normally open strictly alternately, thus allowing both gates to sometimes be open, so transforming the pump into an ion channel. Palytoxin therefore permits electrophysiological analysis of even a single Na͞K pump. We used outside-out patch recording of Xenopus ␣1␤3 Na͞K pumps, which were made ouabain-resistant by point mutation, after expressing them in Xenopus oocytes. Endogenous, ouabain-sensitive, Xenopus ␣1␤3 Na͞K pumps were silenced by continuous exposure to ouabain. We found that side-chain charge of two residues at either end of the ␣ subunit's first extracellular loop, known to make a major contribution to ouabain affinity, strongly influenced conductance of single palytoxin-bound pumpchannels by an electrostatic mechanism. The effects were mimicked by modification of cysteines introduced at those two positions with variously charged methanethiosulfonate reagents. The consequences of these modifications demonstrate that both residues lie in a wide vestibule near the mouth of the pump's ion pathway. Bound ouabain protects the site with the strongest influence on conductance from methanethiosulfonate modification, while leaving the site with the weaker influence unprotected. The results suggest a method for mapping the footprint of bound cardiotonic steroid on the extracellular surface of the Na͞K pump.Na,K-ATPase ͉ palytoxin ͉ single-channel conductance ͉ cysteine modification ͉ electrostatic mechanism

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Section: Discussionmentioning

confidence: 99%

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Artigas

Gadsby

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Tyr 801 Is Not Involved in the Binding of Omeprazole-Cys 815 is the binding site of omeprazole (17) and is reported to be involved in the interaction with SCH 28080 also. The C815T mutant showed a 5-fold higher K i value for SCH 28080 in the NH 4 ϩ -stimulated ATPase activity (22).…”

Section: Construction Of M5 Mutants Of the Gastric Proton Pumpmentioning

confidence: 99%

“…Cysteine residues covalently attached by the active molecules (sulfenamides) of the proton pump inhibitors were identified as Cys 815 and Cys 824 (either in or close to the M6 segment) as well as Cys 323 (in the M3/M4 loop) and Cys 894 (in the M7/M8 loop) (the amino acid positions are given based upon the rabbit enzyme) (17). These cysteine residues themselves are not essential for the function of the proton pump, but the attachment of a bulky drug to the cysteine residues seems to disrupt the conformational change in the proton pump during the catalytic reaction cycles (18).…”

mentioning

confidence: 99%

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

Asano¹,

Yoshida

Yashiro

et al. 2004

Journal of Biological Chemistry

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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...

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“…Rabeprazole, a proton pump inhibitor, has been used for the treatment of H pylori due to its many advantages, including the fact that its pharmacokinetics and pharmacodynamics are not influenced by CYP2C19 genetic polymorphisms [12] . Moreover, its onset of action is faster than other PPIs [13] . Nevertheless, the effects of rabeprazole on the transfer and distribution of amoxicillin in the gastric mucosa and gastric juice are not clear.…”

Section: Introductionmentioning

confidence: 99%

Transfer and distribution of amoxicillin in the rat gastric mucosa and gastric juice and the effects of rabeprazole

Zheng

Bao

et al. 2010

Acta Pharmacol Sin

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Aim: To investigate the distribution of amoxicillin in the gastric juice and gastric mucosa of rats and to investigate the effects of proton pump inhibitor rabeprazole on amoxicillin concentrations in various compartments. Methods: One hundred and sixty anesthetized rats were divided into five groups, and given intravenously different doses of amoxicillin or amoxicillin and rabeprazole. The pH value and volume of gastric juice was aspirated were measured and separated gastric mucosa was homogenized. The concentrations of amoxicillin in the plasma, gastric juice and gastric mucosa were measured by high performance liquid chromatography (HPLC). Results: The maximum concentrations of amoxicillin in gastric juice and gastric mucosa were significantly lower than those in plasma (P<0.001). Concentrations in the glandular stomach mucosa were higher than those in the forestomach mucosa. Rabeprazole did not significantly change the pharmacokinetic parameters of amoxicillin in the plasma and did not alter gastric antibiotic clearance or the gastric transfer fraction of amoxicillin in gastric juice. However, rabeprazole did increase the amoxicillin concentration and pH value in gastric juice and reduced the volume of the gastric juice. Conclusion: Amoxicillin could penetrate the gastric mucosa and achieve therapeutic concentrations at the target site after transfer from the blood to the stomach. Rabeprazole increased the amoxicillin concentration in gastric juice by decreasing the gastric juice volume but did not affect its concentration in blood or gastric mucosa.

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

Cited by 245 publications

References 45 publications

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

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

Transfer and distribution of amoxicillin in the rat gastric mucosa and gastric juice and the effects of rabeprazole

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