The recognition and transport mechanism of ions in the proton pump and its intracellular transport

本课题主要研究胃质子泵对K^+和H^+离子的识别和转运机制及其细胞内转运。泵由α和β亚基组成。首先，我们探索了一种K^+竞争性抑制剂SCH 28080的结合位点，发现前人提出的位点是错误的，泵的α亚基跨膜段6的氨基酸残基参与了SCH 28080的结合。通过质子泵和钠泵的嵌合体研究，我们发现跨膜第6段的氨基酸残基参与了K^+和H^+的识别。跨膜第4段的Glu参与K^+结合和α亚基依赖K^+的构象变化。我们还探讨了泵的β亚基的作用。我们发现位于膜胞外部分外的四个连续氨基酸序列在细胞内具有质子泵目的地信号。该亚基中的碳水化合物链也具有目的地信号，并参与泵的酶活性。β-亚基胞外S-S交联在酶反应中起重要作用。胃壁细胞的顶细胞表面一定具有抵抗细胞自身分泌的强酸的保护机制。作为一种可能的机制，我们发现了一种翻转酶的存在，它可以分泌磷脂酰胆碱和磷脂酰丝氨酸。分泌的磷脂可以作为疏水性衬里并排斥酸。

The main objects of the present research project are to study the recognition and transport mechanisms of K^+ and H^+ ions in the gastric proton pump and its intracellular transport. The pump consists of α-and β-subunits. First, we explored the binding site of SCH 28080 which is a K^+ competitive inhibitor and found that the previously proposed site by others was wrong and the amino acid residues in transmembrane segment 6 of the α subunit of the pump are involved in SCH 28080 binding. From our chimera study between proton pump and Na pump, we found that the amino acid residues in transmembrane segment 6 are involved in K^+ and H^+ recognition. Glu in the transmembrane segment 4 is involved in K^+ binding and the K^+ -dependent conformational change of the α subunit. we also explored the role of the βsubunit of the pump. We found that the four consecutive amino acid sequence located just outside the extracellular part of the membrane has the proton pump destination signal in the cell. Carbohydrate chains in this subunit also has the destination signal and involved in the enzyme activity of the pump. The extracellular S-S cross links in the β-subunit had the important role in the enzyme reaction. The apical cell surface of the gastric parietal cell must, have a protective mechanism against the strong acid secreted by the cell itself. As a possible machinery for this end, we found the presence of a flippase, which can secrete both phosphatidylcholine and phosphatidylserine. The secreted phospholipids may serve as a hydrophopbic lining and rejects acid.