Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli.
Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli.
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DOI：
10.1074/jbc.m113.484071
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发表时间：
2013-08-23
期刊：
影响因子：
--
通讯作者：
Fendler K
中科院分区：
文献类型：
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作者：
Mager T;Braner M;Kubsch B;Hatahet L;Alkoby D;Rimon A;Padan E;Fendler K
背景：大肠杆菌NhaA可以作为Na+/H+交换器中pH调节和转运机制的例子。结果：不同的突变改变Na+和/或H+结合或构象动力学。结论：蛋白质不同片段的突变通过影响其转运机制的不同方面来改变其pH分布。意义：单位点突变和动力学分析揭示了Na+/H+交换机制的决定因素。Na+/H+反向转运蛋白具有明显的pH依赖性，这对它们在真核和原核细胞中的生理功能具有重要意义。在NhaA中，大肠杆菌Na+/H+反向转运蛋白，过去已经描述了调节转运蛋白pH分布的特异性单位点突变。为了阐明这些突变影响NhaA的pH依赖性的机制，选择NhaA变体的动力学的底物依赖性进行电生理学研究，并与动力学模型进行分析。结果表明，突变影响NhaA活性在相当不同的方式，通过改变结合位点的性质或动力学的转运。在第一种情况下，pK和/或KDNa被改变，在第二种情况下，内部和外部开放构象之间的构象转变的速率常数被修改。结果表明，从结合位点远至15-20 μ m的残基可以对构象转变的动力学或对NhaA的结合性质产生显著影响。这些结果的影响NhaA的pH调节机制进行了讨论。
Background: NhaA of Escherichia coli can serve as an example for pH regulation and transport mechanism in Na+/H+ exchangers. Results: Different mutations alter Na+ and/or H+ binding or conformational dynamics. Conclusion: Mutations in different segments of the protein change its pH profile by affecting different aspects of its transport mechanism. Significance: Single site mutations and kinetic analysis reveal determinants of Na+/H+ exchanger mechanism. Na+/H+ antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na+/H+ antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or KDNa are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15–20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed.