Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism
Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism
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DOI：
10.1073/pnas.0902171106
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发表时间：
2009-08-25
影响因子：
11.1
通讯作者：
Vuister, Geerten W.
中科院分区：
文献类型：
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作者：
Hilge, Mark;Aelen, Jan;Vuister, Geerten W.
Na+/Ca~(2+)交换器(NCX)的离子转运调控是通过其胞质内的钙结合区CBD_1和CBD_2实现的。在这里，我们提出了一个NCX的活化和失活的机制，基于核磁共振、等温滴定量热法和小角X射线散射(SAXS)的数据。我们初步确定了代表NCX1中两个主要剪接变体类的CBD2-AD和CBD2-BD的结构。虽然CBD2-AD的脱氧核糖核酸显示了部分无序的钙结合位点，但CBD2-BD的脱氧核糖核酸结合位点是完全无结构的，即使在钙过量的情况下也是如此。钙结合形式和无钙形式之间的显著静电电位差异强烈地表明，CBD1和CBD2中的钙结合部位形成了类似于C-2-结构域的静电开关。对含有CBD1和CBD2的构建物的SAXS分析表明，CBD1通过与钙离子结合而发生构象变化。我们认为，CBD1中的静电开关及其相关的构象变化是交换激活所必需的。CBD1开关对细胞内钙离子的反应受紧密定位的盒式外显子的影响。我们进一步认为，钙离子与CBD2的结合诱导了第二个静电开关，这是减轻Na+依赖的Na+/Ca~(2+)交换失活所必需的。与CBD1不同，CBD2中的静电开关是异构体和剪接变体特有的，并允许量身定做的交换活动。
Regulation of ion-transport in the Na+/Ca2+ exchanger (NCX) occurs via its cytoplasmic Ca2+-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca2+-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca2+-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca2+. Striking differences in the electrostatic potential between the Ca2+-bound and -free forms strongly suggest that Ca2+-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C-2-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca2+-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca2+ is influenced by the closely located cassette exons. We further propose that Ca2+-binding to CBD2 induces a second electrostatic switch, required to alleviate Na+-dependent inactivation of Na+/Ca2+ exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform-and splice variant-specific and allows for tailored exchange activities.