Stable expression of gastric proton pump activity at the cell surface.
Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.
Kimura
Tohru
T
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, 930-0194, Japan.
Yoshida
Ayumi
A
Tabuchi
Yoshiaki
Y
Ikari
Akira
A
Takeguchi
Noriaki
N
Asano
Shinji
S
D001692:Biological Transport; D002478:Cells, Cultured; D005753:Gastric Mucosa / Q000378:metabolism*; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000096:biosynthesis; D010766:Phosphorylation; D017494:Proton Pumps / Q000096:biosynthesis*; D012413:Rubidium / Q000378:metabolism
D008565:Membrane Proteins; D017494:Proton Pumps; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium