|Budget Amount *help
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥2,500,000 (Direct Cost: ¥2,500,000)
The gastric H^+,K^+-ATPase is the proton pump responsible for gastric acid secretion. This pump consists of the catalytic α- and non-catalytic β-subunits. In this research project, we constructed stable cell lines expressing the gastric proton pump, and studied the regulation mechanisms by site-directed mutahenesis.
(1) We constructed three kinds of stable cell lines ; the α-expressing cells, the β-expressing cells, and the α+β-expressing cells. The α-subunit was retained in the intracellular compartment, and no cell surface expression was observed in the absence of the β-subunit. On the other hand, cell surface expression of the β-subunit was observed even in the absence of the α-subunit. Cell surface expression of the α- and β-subunits were observed in the α+β-expressing cells. The α+β-expressing cells represented rubidium (^<86>Rb) and proton transport activities, which were inhibited by proton pump inhibitors.
(2) We also studied the quantity control mechanism of the gastric proton pump in the stable cell lines. The α- and (β-subunits were co-translationally inserted to the ER membrane to form the functional holoenzyme with a stoichiometry of 1 : 1. In this process, unassembled α-subunits were unstable in the cells. They were retained on the ER, modified with chains of polyubiquitin, and degraded by the proteasomes. On the other hand, unassembled β-subunits were more stable, and can travel to the cell surface by itself. We found that the number of functional proton pump, the α/β holoenzyme, was strictly regulated by ubiqutin/proteasome system.
(3) The β-subunit contains three conserved disulfide bond in the ectodomain. We studied the role of these disulfide bonds on the function of the gastric proton pump by site-directed mutagenesis. We found that each disulfide bond was important for the correct assembly between the α- and β-subunits, and their cell surface delivery, and the maintenance of H^+,K^+-ATPase activity.