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

1)胃质子泵在CHO-K1细胞中表达。由于质子泵代替钠泵起作用，这种细胞可以在钠碱存在的情况下生长。质子泵抑制剂奥美拉唑与瓦巴因对质子泵和钠泵的抑制作用共同抑制了细胞的生长。该体系为研究质子泵抑制剂的作用提供了理论依据。2)确定了猪胃高表达Mat-8的初级结构。这种单跨膜蛋白主要表达于粘膜细胞而不表达于壁细胞。因此，了解Mat-8在胃酸（HCl）分泌和粘膜分泌中Cl^-转运中的作用是很有趣的，因为注射Mat-8 mRNA的爪蟾卵母细胞显示出Cl^-通道的活性。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囊泡。我们进一步研究了酵母双杂交系统中与Mat-8相互作用的组分。目前我们发现了几个与Mat-8细胞质结构域相互作用的克隆。我们感兴趣的是这些克隆体编码了什么蛋白质。3)通过在昆虫细胞中表达αβ复合物，测定K^+浓度对EP降解的影响。与猪亚基和大鼠亚基的杂交比较表明，与大鼠β亚基的配合物在低浓度K^+存在下降解EP。β亚基与胃质子泵反应周期中K^+的敏感性有关。

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.