Structure-function analysis of CALX1.1, a Na+-Ca2 exchanger from Drosophila -: Mutagenesis of ionic regulatory sites
Structure-function analysis of CALX1.1, a Na+-Ca2 exchanger from Drosophila -: Mutagenesis of ionic regulatory sites
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DOI：
10.1074/jbc.273.21.12981
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发表时间：
1998-05-22
影响因子：
4.8
通讯作者：
Hryshko, LV
中科院分区：
文献类型：
--
作者：
Dyck, C;Maxwell, K;Hryshko, LV
细胞质Na+和Ca 2+调节Na+-Ca 2+交换蛋白的活性，除了作为转运离子，并且已经鉴定了犬心脏Na+-Ca 2+交换蛋白NCX1.1中参与这些过程的蛋白质区域。尽管与Na+和Ca+依赖性调节相关的蛋白质区域在克隆的Na+-Ca 2+交换剂中高度保守，但不知道NCX1.1的结构-功能关系特征是否适用于任何其他交换剂。因此，我们研究了来自果蝇的Na+-Ca 2+交换器CALX1.1的结构-功能关系，CALX1.1在该蛋白家族的特征成员中是独特的，因为μ M水平的Ca 1（2+）抑制交换电流。在非洲爪蟾卵母细胞中表达了野生型和突变型CALX1.1交换蛋白，并使用巨大切除贴片技术进行电生理学表征。D516 V和D5501）或Ca 1（2+）不能抑制Na+-Ca 2+交换活性（即G555 P）。类似地，与NCX1.1一样，CALX1.1的推定XIP区域内的突变导致两种不同的表型：Na-1（+）依赖性失活的加速（即K306 Q）和消除（即Delta 310-313）。这些结果表明，Ca-i（2+）结合位点和XIP区域的各自调节作用在CALX1.1和NCX1.1之间是保守的，尽管对Ca-i（2+）的反应相反。我们使用CALX1.1和NCX1.1的嵌合构建体来扩展这些发现，以确定Ca-i（2+）调节表型的功能互换是否可行。有一个嵌合体（即，CALX：NCX：CALX），将来自NCX1.1的大胞内环的193个氨基酸的片段替换为相应的CALX 1.1的177个氨基酸的片段，导致由Ca-1（2+）刺激的交换剂。该结果表明，NCX1.1的调节性Ca-1（2+）结合位点保留了在CALX1.1亲本转运蛋白中的功能，并且取代的区段含有转导Ca-1（2+）结合信号所需的一些氨基酸序列。
Cytoplasmic Na+ and Ca2+ regulate the activity of Na+-Ca2+ exchange proteins, in addition to serving as the transported ions, and protein regions involved in these processes have been identified for the canine cardiac Na+-Ca2+ exchanger, NCX1.1. Although protein regions associated with Na-i(+)- and Ca-i(2+)-dependent regulation are highly conserved among cloned Na+-Ca2+ exchangers, it is unknown whether or not the structure-function relationships characteristic of NCX1.1 apply to any other exchangers. Therefore, we studied structure-function relationships in a Na+-Ca2+ exchanger from Drosophila, CALX1.1, which is unique among characterized members of this family of proteins in that mu M levels of Ca-i(2+) inhibit exchange current. Wild-type and mutant CALX1.1 exchangers were expressed in Xenopus oocytes and characterized electrophysiologically using the giant excised patch technique, Mutations within the putative regulatory Ca-i(2+) binding site of CALX1.1, like corresponding alterations in NCX1.1, led to reduced ability (i,e. D516V and D5501) or inability (i.e. G555P) of Ca-i(2+) to inhibit Na+-Ca2+ exchange activity. Similarly, mutations within the putative XIP region of CALX1.1, as in NCX1.1, led to two distinct phenotypes: acceleration (i.e. K306Q) and elimination (i.e. Delta 310-313) of Na-i(+)-dependent inactivation. These results indicate that the respective regulatory roles of the Ca-i(2+) binding site and XIP region are conserved between CALX1.1 and NCX1.1, despite opposite responses to Ca-i(2+), We extended these findings using chimeric constructs of CALX1.1 and NCX1.1 to determine whether or not functional interconversion of Ca-i(2+) regulatory phenotypes was feasible. With one chimera (i,e. CALX:NCX:CALX), substitution of a 193-amino acid segment, from the large intracellular loop of NCX1.1, for the corresponding 177-amino acid segment of CALX1.1 led to an exchanger that was stimulated by Ca-i(2+). This result indicates that the regulatory Ca-i(2+) binding site of NCX1.1 retains function in a CALX1.1 parent transporter and that the substituted segment contains some of the amino acid sequence(s) required for transduction of the Ca-i(2+) binding signal.