Acidic residues involved in cation and substrate interactions in the Na+/dicarboxylate cotransporter, NaDC-1
Acidic residues involved in cation and substrate interactions in the Na+/dicarboxylate cotransporter, NaDC-1
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DOI：
10.1021/bi990076b
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发表时间：
1999-06-08
期刊：
影响因子：
2.9
通讯作者：
Pajor, AM
中科院分区：
文献类型：
--
作者：
Griffith, DA;Pajor, AM
通过定点突变和爪蟾卵母细胞中的表达研究了酸性氨基酸残基在阳离子中的作用：Na+/二羧酸协同转运蛋白NaDC-1的识别和选择性。发现检测的4个残基（Asp-52、Glu-74、Glu-101和Glu-332）对转运活性不重要。然而，替换的Asp-373和Glu-475，在跨膜结构域M8和M9中发现的保守残基，分别改变运输动力学。替换的Asp-373与：丙氨酸，谷氨酸，天冬酰胺，和Gin导致在钠的亲和力和阳离子选择性的变化，在NaDC-1，这表明在这个位置的羰基氧可能发挥作用的拓扑组织的阳离子结合位点。与此相反，取代Glu-475导致运输活动和运输动力学的变化显着减少。用Gin取代导致具有增加的底物和钠亲和力的转运蛋白，而E475 D突变体是无活性的。E475 A突变体似乎具有差的钠结合。E475 A突变体中的底物诱导电流表现出强烈的电压依赖性，并且在-30 mV下观察到电流的逆转。结果表明Glu-475可能在阳离子结合中起作用，也可能在介导阴离子通道活性中起作用;值得注意的是，Asp-373和Glu-475的突变都影响NaDC-1中琥珀酸的Km，这表明这些残基在决定对底物和阳离子的亲和力方面具有双重作用。我们提出，至少有一个阳离子结合位点和底物结合位点在NaDC-1的羧基末端部分靠近在一起，因此跨膜结构域M8和M9是形成易位途径的候选结构。
The role of acidic amino acid residues in cation:recognition and selectivity by the Na+/dicarboxylate cotransporter, NaDC-1, was investigated by site-directed mutagenesis and expression in Xenopus oocytes. Four of the residues tested, Asp-52, Glu-74, Glu-101, and Glu-332, were found to be unimportant, for transport activity. However, substitutions of Asp-373 and Glu-475, conserved residues found in transmembrane domains M8 and M9, respectively, altered transport kinetics. Replacements of Asp-373 with:Ala, Glu, Asn, and Gin resulted in changes in sodium affinity and cation selectivity in NaDC-1, indicating that the carbonyl oxygen at this position may play a role in the topological organization of the;cation-binding site. In contrast, substitutions of Glu-475 led to dramatic reductions in transport activity and changes in transport kinetics. Substitution with Gin led to a transporter with increased substrate and sodium affinity, while the E475D mutant was inactive. The E475A mutant appeared to have poor sodium binding. Substrate-induced currents in the E475A mutant exhibited a strong voltage dependence, and a reversal of the current was seen at -30 mV. The results suggest that Glu-475 may play a role in cation binding and possibly also in mediating anion channel activity; Remarkably, mutations of both Asp-373 and Glu-475 affected the K-m for succinate in NaDC-1, suggesting' dual roles for these residues indetermining the affinity for substrate and cations. We propose that at least one of the cation-binding sites and the substrate-binding site are close together in the carboxy-terminal: portion of NaDC-1, and thus transmembrane domains M8 and;M9 are candidate structures for the formation of the translocation pathway.