Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon 16419, South Korea.
Biochim Biophys Acta Bioenerg. 2019 Mar 1;1860(3):189-200. doi: 10.1016/j.bbabio.2018.11.015. Epub 2018 Nov 8.
The cell membrane (NCX) and mitochondrial (NCLX) Na/Ca exchangers control Ca homeostasis. Eleven (out of twelve) ion-coordinating residues are highly conserved among eukaryotic and prokaryotic NCXs, whereas in NCLX, nine (out of twelve) ion-coordinating residues are different. Consequently, NCXs exhibit high selectivity for Na and Ca, whereas NCLX can exchange Ca with either Na or Li. However, the underlying molecular mechanisms and physiological relevance remain unresolved. Here, we analyzed the NCX_Mj-derived mutant NCLX_Mj (with nine substituted residues) imitating the ion selectivity of NCLX. Site-directed fluorescent labeling and ion flux assays revealed the nearly symmetric accessibility of ions to the extracellular and cytosolic vestibules in NCLX_Mj (K = 0.8-1.4), whereas the extracellular vestibule is predominantly accessible to ions (K = 0.1-0.2) in NCX_Mj. HDX-MS (hydrogen-deuterium exchange mass-spectrometry) identified symmetrically rigidified core helix segments in NCLX_Mj, whereas the matching structural elements are asymmetrically rigidified in NCX_Mj. The HDX-MS analyses of ion-induced conformational changes and the mutational effects on ion fluxes revealed that the "Ca-site" (S) of NCLX_Mj binds Na, Li, or Ca, whereas one or more additional Na/Li sites of NCLX_Mj are incompatible with the Na sites (S and S) of NCX_Mj. Thus, the replacement of ion-coordinating residues in NCLX_Mj alters not only the ion selectivity of NCLX_Mj, but also the capacity and affinity for Na/Li (but not for Ca) binding, bidirectional ion-accessibility, the response of the ion-exchange to membrane potential changes, and more. These structure-controlled functional features could be relevant for differential contributions of NCX and NCLX to Ca homeostasis in distinct sub-cellular compartments.
细胞膜（NCX）和线粒体（NCLX）Na/Ca 交换器控制 Ca 离子的动态平衡。真核生物和原核生物的 NCX 中，有 11 个（共 12 个）离子配位残基高度保守，而在 NCLX 中，有 9 个（共 12 个）离子配位残基不同。因此，NCX 对 Na 和 Ca 具有高选择性，而 NCLX 可以用 Na 或 Li 交换 Ca。然而，其潜在的分子机制和生理相关性仍未解决。在这里，我们分析了模拟 NCLX 离子选择性的源自 NCX_Mj 的突变体 NCLX_Mj（有 9 个取代残基）。定点荧光标记和离子通量测定显示，NCLX_Mj 中离子对胞外和胞质小室几乎具有相同的可及性（K 值为 0.8-1.4），而在 NCX_Mj 中，胞外小室主要可被离子进入（K 值为 0.1-0.2）。氢氘交换质谱（HDX-MS）鉴定出 NCLX_Mj 中对称刚性核心螺旋片段，而在 NCX_Mj 中对应的结构元件则是非对称刚性的。离子诱导构象变化的 HDX-MS 分析以及对离子通量的突变效应表明，NCLX_Mj 的“Ca 结合位点”（S）可以结合 Na、Li 或 Ca，而 NCLX_Mj 的一个或多个额外的 Na/Li 结合位点与 NCX_Mj 的 Na 结合位点（S 和 S）不兼容。因此，NCLX_Mj 中离子配位残基的替换不仅改变了 NCLX_Mj 的离子选择性，还改变了其对 Na/Li（而非 Ca）结合的容量和亲和力、离子的双向可及性、离子交换对膜电位变化的响应以及更多的功能。这些结构控制的功能特征可能与 NCX 和 NCLX 在不同亚细胞区室对 Ca 离子动态平衡的不同贡献有关。
