Mechanism, Intracellular Transport and Physiological Function of P-type ATPase

1)CHO-K1细胞表达胃质子泵。这种细胞可以在哇巴因存在下生长，因为质子泵代替钠泵发挥作用。质子泵抑制剂奥美拉唑与哇巴因合用可抑制细胞生长，其抑制作用与其分别抑制质子泵和钠泵有关。该系统为研究质子泵转运体的作用奠定了基础。2）测定了猪胃中高表达Mat-8蛋白的一级结构。这种单一的跨膜蛋白主要在粘液细胞中表达，而在壁细胞中不表达。因此，了解Mat-8在胃酸（HCl）分泌和粘液分泌中的Cl^-转运中的作用是很有趣的，因为注射Mat-8 mRNA的非洲爪蟾卵母细胞显示Cl^-通道活性。细胞分布检测表明，Mat-8与DS-Red的融合蛋白定位于内质网和胞质小泡。我们进一步利用酵母双杂交系统寻找与Mat-8相互作用的组分。目前，我们发现了几个与Mat-8胞质结构域相互作用的克隆。我们对这些克隆编码的蛋白质很感兴趣。3）通过昆虫细胞中表达的αβ复合物来确定K^+浓度对EP降解的影响。与猪和大鼠β亚基的杂交体的比较表明，与大鼠β亚基的复合物在较低浓度的K^+存在下降解EP。β亚基在胃质子泵反应周期中对K^+敏感性起作用。

1) The gastric proton pump was expressed in CHO-K1 cells. Such cell could grow in the presence of ouabain, since proton pump functions in place of sodium pump. Proton pump inhibitor, omeprazol, together with ouabain inhibited the cell growth, consisting with their inhibitory effects on proton pump and sodium pump, respectively. This system would be useful for study the effect of proton pump inhibitor.2) Primary structure of pig Mat-8 that highly expresses in stomach was determined. This single transmembrane protein mainly expresses in mucous cells but not parietal cells. Thus it is interesting to know how Mat-8 functions in Cl^- transport in gastric acid (HCl) secretion and mucous secretion, since Xenopus oocyte injected with Mat-8 mRNA showed Cl^- channel activity. Examination of cellular distribution demonstrated that the fusion protein of Mat-8 with DS-Red localized in endoplasmic reticulum and cytoplasmic vesicles. We further studied to find the component(s) interacting with Mat-8 by yeast two-hybrid system. At present we found several clones that interact with cytoplasmic domain of Mat-8. We are interested in what proteins are encoded by these clones.3) Effect of K^+ concentration on the degradation of EP was determined with the αβ complex expressed in insect cells. Comparison of hybrids with pig and rat subunits demonstrated that the complex with rat β subunit degraded EP in the presence of lower concentration of K^+. The β subunit has a role in K^+ sensitivity in the reaction cycle of the gastric proton pump.