The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity
The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity
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DOI：
10.1074/jbc.m003788200
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发表时间：
2000-12-08
影响因子：
4.8
通讯作者：
Shigekawa, M
中科院分区：
文献类型：
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作者：
Iwamoto, T;Uehara, A;Shigekawa, M
我们通过半胱氨酸替代扫描分析和定点突变体的动力学分析，研究了包含保守内部α重复序列区域的Na⁺/Ca²⁺交换体NCX1的膜拓扑结构以及残基的功能重要性。结果表明，α -1重复序列以及包含α -α重复序列及其紧邻的C末端片段的区域都含有折返环结构域，每个结构域相对于膜的取向相反。我们发现，α重复序列折返结构域中的6个残基（包括Asn - 125以及保守的天冬氨酸Asp - 130、Asp - 825和Asp - 829）的单个或多个突变使交换体对细胞外Ca²⁺的表观亲和力降低多达9倍。相反，三联半胱氨酸突变D130C/D825C/D829C并不影响交换电流的电流 - 电压（I - V）关系。用不同的巯基修饰剂进行的半胱氨酸可及性扫描表明，当用外部探针检测时，N125C、D130C和D825C可能位于膜内一个受限的水空间中，只有离子能够进入，尽管先前已表明在交换反应过程中N125C和D825C可从内部接近。结果表明，α重复序列中的这些折返结构域可能参与交换体中离子转运途径的形成，其中一些天冬氨酸可能位于该途径上或靠近它。
We examined the membrane topology and functional importance of residues in regions of the Na+/Ca2+ exchanger NCX1 encompassing the conserved internal a repeats by substituted cysteine scanning analysis and kinetic analysis of site directed mutants. The results suggest that both the alpha -1 repeat and a region encompassing the a-a repeat and its immediately C-terminal segment contain reentrant loop domains, each oriented in an opposite direction with respect to the membrane, We found that single or multiple mutations of six residues including Asn-125 and conserved aspartates Asp-130, Asp-825, and Asp-829 in the a repeat reentrant domains reduce the apparent affinity of the exchanger for extracellular Ca2+ by up to g-fold, In contrast, the triple cysteine mutation D130C/D825C/D829C did not influence the current-voltage (I-V) relationship of the exchange current. Cysteine accessibility scanning with different thiol modifiers suggested that N125C, D130C, and D825C may be located in a restricted aqueous space in the membrane accessible only to ions when examined with external probes, although N125C and D825C were previously shown to be internally accessible during exchange reaction. The results suggest that these reentrant domains in the a repeats may participate in the formation of the ion transport pathway in the exchanger with some of the aspartates possibly lining it or located close to it.