Qiu Z, Nicoll D A, Philipson K D
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1760, USA.
J Biol Chem. 2001 Jan 5;276(1):194-9. doi: 10.1074/jbc.M005571200.
In a revised topological model of the cardiac Na(+)-Ca(2+) exchanger, there are nine transmembrane segments (TMSs) and two possible re-entrant loops (Nicoll, D. A., Ottolia, M., Lu, Y., Lu, L., and Philipson, K. D. (1999) J. Biol. Chem. 274, 910-917; Iwamoto, T., Nakamura, T. Y., Pan, Y., Uehara, A., Imanaga, I., and Shigekawa, M. (1999) FEBS Lett. 446, 264-268). The TMSs form two clusters separated by a large intracellular loop between TMS5 and TMS6. We have combined cysteine mutagenesis and oxidative cross-linking to study proximity relationships of TMSs in the exchanger. Pairs of cysteines were reintroduced into a cysteine-less exchanger, one in a TMS in the NH(2)-terminal cluster (TMSs 1-5) and the other in a TMS in the COOH-terminal cluster (TMSs 6-9). The mutant exchanger proteins were expressed in HEK293 cells, and disulfide bond formation between introduced cysteines was analyzed by gel mobility shifts. Western blots showed that S117C/V804C, A122C/Y892C, A151C/T815C, and A151C/A821C mutant proteins migrated at 120 kDa under reducing conditions and displayed a partial mobility shift to 160 kDa under nonreducing conditions. This shift indicates the formation of a disulfide bond between these paired cysteine residues. Copper phenanthroline and the cross-linker N', N'-o-phenylenedimaleimide enhanced the mobility shift to 160 kDa. Our data suggest that TMS7 is close to TMS3 near the intracellular side of the membrane and is in the vicinity of TMS2 near the extracellular surface. Also, TMS2 must adjoin TMS8. This initial packing model of the exchanger brings two functionally important domains in the exchanger, the alpha 1 and alpha 2 repeats, close to each other.
在心脏钠钙交换体的修正拓扑模型中，有九个跨膜片段（TMSs）和两个可能的折返环（尼科尔，D.A.，奥托利亚，M.，陆，Y.，陆，L.，和菲利普森，K.D.（1999年）《生物化学杂志》274卷，910 - 917页；岩本，T.，中村，T.Y.，潘，Y.，上原，A.，今永，I.，和重川，M.（1999年）《欧洲生物化学学会联合会快报》446卷，264 - 268页）。这些跨膜片段形成两个簇，由跨膜片段5和跨膜片段6之间的一个大的细胞内环分隔。我们结合了半胱氨酸诱变和氧化交联来研究交换体中跨膜片段的邻近关系。将半胱氨酸对重新引入无半胱氨酸的交换体中，一个位于氨基末端簇的跨膜片段（跨膜片段1 - 5）中，另一个位于羧基末端簇的跨膜片段（跨膜片段6 - 9）中。突变的交换体蛋白在HEK293细胞中表达，并通过凝胶迁移率变化分析引入的半胱氨酸之间二硫键的形成。蛋白质免疫印迹显示，S117C/V804C、A122C/Y892C、A151C/T815C和A151C/A821C突变蛋白在还原条件下迁移至120 kDa，在非还原条件下部分迁移至160 kDa。这种迁移变化表明这些成对的半胱氨酸残基之间形成了二硫键。邻菲罗啉铜和交联剂N'，N'-邻苯二甲酰亚胺增强了迁移至160 kDa的变化。我们的数据表明，跨膜片段7在膜的细胞内侧靠近跨膜片段3，在细胞外侧靠近跨膜片段2。此外，跨膜片段2必须与跨膜片段8相邻。交换体的这种初始堆积模型使交换体中两个功能重要的结构域，即α1和α2重复序列，彼此靠近。
