Theoretical Molecular Biophysics Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
Membrane Transport Biophysics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.
J Gen Physiol. 2018 Jan 2;150(1):51-65. doi: 10.1085/jgp.201711897. Epub 2017 Dec 13.
Intracellular Ca signals control a wide array of cellular processes. These signals require spatial and temporal regulation of the intracellular Ca concentration, which is achieved in part by a class of ubiquitous membrane proteins known as sodium-calcium exchangers (NCXs). NCXs are secondary-active antiporters that power the translocation of Ca across the cell membrane by coupling it to the flux of Na in the opposite direction, down an electrochemical gradient. Na and Ca are translocated in separate steps of the antiport cycle, each of which is thought to entail a mechanism whereby ion-binding sites within the protein become alternately exposed to either side of the membrane. The prokaryotic exchanger NCX_Mj, the only member of this family with known structure, has been proposed to be a good functional and structural model of mammalian NCXs; yet our understanding of the functional properties of this protein remains incomplete. Here, we study purified NCX_Mj reconstituted into liposomes under well-controlled experimental conditions and demonstrate that this homologue indeed shares key functional features of the NCX family. Transport assays and reversal-potential measurements enable us to delineate the essential characteristics of this antiporter and establish that its ion-exchange stoichiometry is 3Na:1Ca Together with previous studies, this work confirms that NCX_Mj is a valid model system to investigate the mechanism of ion recognition and membrane transport in sodium-calcium exchangers.
细胞内 Ca 信号控制着广泛的细胞过程。这些信号需要对细胞内 Ca 浓度进行时空调节，这部分是通过一类普遍存在的膜蛋白来实现的，这些蛋白被称为钠钙交换器（NCXs）。NCXs 是一种次级主动转运体，通过将 Ca 与 Na 的反向通量耦合，利用电化学梯度来驱动 Ca 穿过细胞膜的转运。Na 和 Ca 在反向转运周期的单独步骤中被转运，每个步骤都被认为涉及一种机制，即蛋白内的离子结合位点交替暴露在膜的两侧。原核交换器 NCX_Mj 是该家族中唯一具有已知结构的成员，它被提议作为哺乳动物 NCXs 的良好功能和结构模型；然而，我们对该蛋白的功能特性的理解仍然不完整。在这里，我们在严格控制的实验条件下研究了重新构建到脂质体中的纯化 NCX_Mj，并证明这种同源物确实具有 NCX 家族的关键功能特征。转运测定和反转电位测量使我们能够描绘出这种反向转运体的基本特征，并确定其离子交换计量比为 3Na:1Ca。结合以前的研究，这项工作证实了 NCX_Mj 是研究钠钙交换器中离子识别和膜转运机制的有效模型系统。
