Ren Xiaoyan, Nicoll Debora A, Philipson Kenneth D
Department of Physiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1760, USA.
J Biol Chem. 2006 Aug 11;281(32):22808-14. doi: 10.1074/jbc.M604753200. Epub 2006 Jun 19.
The cardiac Na+-Ca2+ exchanger (NCX1) is a membrane protein that extrudes Ca2+ from cells using the energy of the Na+ gradient and is a key protein in regulating intracellular Ca2+ and contractility. Based on the current topological model, NCX1 consists of nine transmembrane segments (TMSs). The N-terminal five TMSs are separated from the C-terminal four TMSs by a large intracellular loop. Cysteine 768 is modeled to be in TMS 6 close to the intracellular surface. In this study, the proximity of TMS 6 to TMSs 1 and 2 was examined. Insect High Five cells were transfected with cDNAs encoding mutant NCX1 proteins. Each mutant contained cysteine 768 and an introduced cysteine in TMS 1 or 2. Cross-linking between cysteines was determined after reaction with thiol-specific cross-linkers containing spacer arms of 6.5-12 A. The data indicate that residues in TMSs 1 and 2 are close to cysteine 768 in TMS 6. Cysteine 768 cross-linked with residues at both ends of TMSs 1 and 2 and is likely located toward the middle of TMS 6. Based on these results, we present an expanded helix-packing model for NCX1.
心脏钠钙交换体（NCX1）是一种膜蛋白，它利用钠离子梯度的能量将钙离子排出细胞，是调节细胞内钙离子和收缩性的关键蛋白。根据当前的拓扑模型，NCX1由九个跨膜片段（TMSs）组成。N端的五个TMSs通过一个大的细胞内环与C端的四个TMSs分隔开。半胱氨酸768被模拟定位在靠近细胞内表面的TMS 6中。在本研究中，检测了TMS 6与TMSs 1和2的接近程度。用编码突变型NCX1蛋白的cDNA转染昆虫High Five细胞。每个突变体在TMS 1或2中含有半胱氨酸768和一个引入的半胱氨酸。在用含有6.5 - 12 Å间隔臂的硫醇特异性交联剂反应后，测定半胱氨酸之间的交联情况。数据表明，TMSs 1和2中的残基与TMS 6中的半胱氨酸768接近。半胱氨酸768与TMSs 1和2两端的残基交联，并且可能位于TMS 6的中部。基于这些结果，我们提出了一个扩展的NCX1螺旋堆积模型。
