Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na+/H+ antiporter of Escherichia coli
Trans membrane domain IV is involved in ion transport activity and pH regulation of the NhaA-Na+/H+ antiporter of Escherichia coli
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DOI：
10.1021/bi011655v
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发表时间：
2002-01-15
期刊：
影响因子：
2.9
通讯作者：
Padan, E
中科院分区：
文献类型：
--
作者：
Galili, L;Rothman, A;Padan, E
我们之前已经证明 NhaA 的活性受 pH 调节，并发现突变会显着影响速率的 pH 依赖性，但不会影响 NhaA 的 K-m（对于 Na+ 和 Li+）。在目前的工作中，我们发现螺旋 IV 参与 NhaA 的离子易位和 pH 调节。发现两种新型 NhaA 突变体聚集在跨膜片段 (TMS) IV 中：一种类型（D133C、T132C 和 P129L）影响 NhaA 与阳离子的表观 K-m，但对反向转运蛋白的 pH 谱没有显着影响；当在饱和 Na+ 浓度下测量这些突变体的活性时，没有发现 pH 曲线发生变化。相反，发现其他类型的突变（A127V 和 A127T）会影响 NhaA 速率的 K-m 和 pH 依赖性，无论是否在饱和 Na+ 浓度下进行测试。这些结果意味着参与 NhaA 离子易位的残基可能 (A127) 也可能不 (D133、T132 和 P129) 与影响反向转运蛋白 pH 响应的残基重叠。所有突变体都聚集在假定的α螺旋IV的N端一半，一种类型在一个面上，另一种在相反的面上。 Cys可达性测试表明，虽然D133C位于TMS IV中部，但它受到N-乙基马来酰亚胺的抑制并暴露于细胞质。
We have previously shown that the activity of NhaA is regulated by pH and found mutations that affect dramatically the pH dependence of the rate but not the K-m (for Na+ and Li+) of NhaA. In the present work, we found that helix IV is involved both in ion translocation as well as in pH regulation of NhaA. Two novel types of NhaA mutants were found clustered in trans membrane segment (TMS) IV: One type (D133C, T132C, and P129L) affects the apparent K-m of NhaA to the cations with no significant effect on the pH profile of the antiporter; no shift of the pH profile was found when the activity of these mutants was measured at saturating Na+ concentration. In contrast, the other type of mutations (A127V and A127T) was found to affect both the K-m and the pH dependence of the rate of NhaA whether tested at saturating Na+ concentration or not. These results imply that residues involved in the ion translocation of NhaA may (A127) or may not (D133, T132, and P129) overlap with those affecting the pH response of the antiporter. All mutants cluster in the N-terminal half of the putative a-helix IV, one type on one face, the other on the opposite. Cys accessibility test demonstrated that although D133C is located in the middle of TMS IV, it is inhibited by N-ethylmaleimide and is exposed to the cytoplasm.