Novel physiological function of CLC-5 chloride channel associated with gastric proton pump

在胃壁细胞中，质子是由胃质子泵(H.K-ATPase)主动分泌的，但目前尚不清楚是什么分子参与了顶端氯离子转运以促进HCl的分泌。虽然ClC-2被认为是心尖Cl~-通道的候选者，但我们发现在胃壁细胞中没有ClC-2蛋白的表达。有趣的是，ClC-5在壁细胞中表达。在本研究中，我们研究了ClC-5在壁细胞中的生理功能。我们发现ClC-5蛋白与胃H，K-ATPase共定位于壁细胞的管泡和顶膜。用抗H，K-ATPase抗体进行免疫沉淀，证实了猪胃小泡中CLC-5与H，K-ATPase的相关性。我们成功地在HEK293细胞中建立了四环素调控的ClC-5稳定表达细胞，稳定表达H，K-ATPaseα-和β-亚基。经四环素处理后，CLC-5在细胞膜和细胞器中均有表达，而未经四环素处理的细胞中CLC-5无明显表达。CLC-5与胃H，K-ATPase共定位于四环素处理的细胞膜。在这些细胞中，ClC-5的表达显著增加了H，K-ATPase的活性、86&Gt；Rb+转运和H，K-ATPase的磷酸化水平(EP水平)。在细胞表面H，K-ATPaseα亚基的生物素化中，我们发现ClC-5的表达不影响质膜H，K-ATPase的表达水平。这些结果表明ClC-5是一种新的胃H，K-ATPase上调因子。

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.