| Budget Amount *help |
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In gastric parietal cells, protons are actively secreted by gastric proton pump (H.K-ATPase), but it has not been established what molecule contributes to apical Cl^- transport for HCl secretion. Although CLC-2 was suggested to be a candidate for the apical Cl^- channel, we found here that no CLC-2 protein is expressed in the gastric parietal cells. Interestingly, CLC-5 was found to be expressed in the parietal cells.
In the present study, we studied about the physiological function of CLC-5 in the parietal cells. We found that CLC-5 protein is co-localized with gastric H,K-ATPase in the tubulovesicles and the apical membrane of the parietal cells. Immunoprecipitation using the anti-H,K-ATPase antibody showed the association between CLC-5 and H,K-ATPase in isolated hog gastric vesicles. We succeeded to establish the tetracycline-regulated stable expression of CLC-5 in the HEK293 cells that stably express H,K-ATPase α- and β-subunits. When the cells were treated with tetracycline, CLC-5 was expressed in the plasma membrane and intracellular organelles, while no significant expression of CLC-5 was observed in the cells treated without tetracycline. CLC-5 was co-localized with gastric H,K-ATPase in the plasma membrane of the tetracycline-treated cells. In these cells, the expression of CLC-5 significantly increased the activity of H,K-ATPase, ^<86>Rb^+ transport and phosphorylation level (EP level) of the H,K-ATPase. In the cell surface biotinylation of H,K-ATPase α-subunit, we found that the expression of CLC-5 did not affect the expression level of H,K-ATPase in the plasma membrane. These results suggest that CLC-5 acts as a novel up-regulator of gastric H,K-ATPase.
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