A Hybrid between Na+,K+-ATPase and H+,K+-ATPase Is Sensitive to Palytoxin, Ouabain, and SCH 28080

Robert A Farley,Silvia Schreiber,Shyang-Guang Wang,Georgios Scheiner-Bobis

doi:10.1074/jbc.m008784200

Robert A Farley, Silvia Schreiber + Show 2 more

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https://doi.org/10.1074/jbc.m008784200

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Abstract

Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.

Highlights

Na؉,K؉-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H؉,K؉-ATPase
A chimeric polypeptide consisting of the rat sodium pump ␣3 subunit with the peptide Gln905–Val930 of the gastric proton pump ␣ subunit substituted in place of the original Asn886–Ala911 sequence was expressed together with the gastric ␤ subunit in the yeast Saccharomyces cerevisiae
The work of many investigators has been focused on the identification of amino acids or peptides of the proton and sodium pumps involved in interaction with omeprazole and SCH 28080, or ouabain and related cardiac glycosides, respectively

Summary

Introduction

Na؉,K؉-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H؉,K؉-ATPase. The ␤ subunits are 30% identical and form a single membrane span with the amino terminus on the extracellular side of the membrane Despite this limited identity of the primary structure, the tertiary structure of both types of ␤ subunits must be very similar, since the proton pump ␤ subunit was shown in numerous investigations to form functional complexes with the ␣ subunit of the sodium pump when these proteins were expressed together (10 –14). Analysis of a series of sodium pump mutants helped to identify several amino acids involved in the recognition of ouabain (25–28), but little is known about amino acids or peptides of the gastric proton pump involved in the recognition of omeprazole or SCH 28080 Amino acids in both the aminoand carboxyl-terminal halves of the ␣ subunit of gastric Hϩ,KϩATPase have been suggested to participate in the binding sites for these inhibitors. Since omeprazole binding to the proton pump can be inhibited by SCH 28080 (31), this result raises the possibility that amino acids within the COOHterminal half of the gastric pump ␣ subunit might partic-

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