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

1)胃质子泵在CHO-K1细胞中表达。这种细胞可以在哇巴因存在的情况下生长，因为质子泵取代了钠泵发挥作用。质子泵抑制剂奥美拉唑和哇巴因对细胞生长的抑制作用分别与其对质子泵和钠泵的抑制作用相一致。2)确定了在胃中高效表达的猪Mat-8的一级结构。这种单一的跨膜蛋白主要在粘液细胞中表达，而不在壁细胞中表达。因此，了解Mat-8在胃酸(HCl)分泌和粘液分泌中的作用是很有意义的，因为注射了Mat-8mRNA的非洲爪哇卵母细胞具有氯离子通道活性。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细胞质小泡中。我们进一步研究了利用酵母双杂交系统寻找与MAT-8相互作用的成分(S)。目前，我们发现了几个与Mat-8胞质结构域相互作用的克隆。我们感兴趣的是这些克隆编码了什么蛋白质。3)通过在昆虫细胞中表达的αβ复合体测定了K^浓度对EP降解的影响。与猪和大鼠亚单位杂交的比较表明，与大鼠β亚单位形成的复合体在较低浓度的K~+存在下能降解EP。在胃质子泵的反应周期中，β亚基在K？敏感性中起作用。

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.