Cai Xinjiang, Zhang Kathy, Lytton Jonathan
Cardiovascular Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2002 Dec 13;277(50):48923-30. doi: 10.1074/jbc.M208818200. Epub 2002 Oct 10.
In this study we have examined the roles of endogenous cysteine residues in the rat brain K(+)-dependent Na(+)/Ca(2+) exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation system. We speculated that Cys-614 and Cys-666 might form an extracellular disulfide bond that stabilized protein structure. Such an arrangement would place the C terminus of the exchanger outside the cell, contrary to the original topological model. This hypothesis was tested by adding a hemagglutinin A epitope to the C terminus of the protein. The hemagglutinin A epitope could be recognized with a specific antibody without permeabilization of the cell membrane, supporting an extracellular location for the C terminus. Additionally, the exchanger molecule could be labeled with biotin maleimide only following extracellular application of beta-mercaptoethanol. Surprisingly, mutation of Cys-395, located in the large intracellular loop, to Ala, prevented reduction-dependent labeling of the protein. The activity of wild-type exchanger, but not the Cys-395 --> Ala mutant, was stimulated after application of beta-mercaptoethanol. Co-immunoprecipitation experiments demonstrated self-association between wild-type and FLAG-tagged exchanger proteins that could not be inhibited by Cys-395 --> Ala mutation. These results suggest that NCKX2 associates as a dimer, an interaction that does not require, but may be stabilized by, a disulfide linkage through Cys-395. This linkage, perhaps by limiting protein mobility along the dimer interface, reduces the transport activity of NCKX2.
在本研究中，我们通过定点诱变研究了内源性半胱氨酸残基在大鼠脑钾离子依赖性钠/钙交换蛋白NCKX2中的作用。我们发现，将半胱氨酸-614或半胱氨酸-666突变为丙氨酸会抑制该交换蛋白在HEK-293细胞中的表达，但在体外翻译系统中则不会。我们推测，半胱氨酸-614和半胱氨酸-666可能形成一个细胞外二硫键，从而稳定蛋白质结构。这种排列会使交换蛋白的C末端位于细胞外，这与原来的拓扑模型相反。通过在该蛋白的C末端添加一个血凝素A表位对这一假设进行了验证。血凝素A表位可以被特异性抗体识别，而无需使细胞膜通透，这支持了C末端位于细胞外的定位。此外，只有在细胞外应用β-巯基乙醇后，交换蛋白分子才能被生物素马来酰亚胺标记。令人惊讶的是，位于大的细胞内环中的半胱氨酸-395突变为丙氨酸后，会阻止该蛋白的还原依赖性标记。应用β-巯基乙醇后，野生型交换蛋白的活性受到刺激，而半胱氨酸-395突变为丙氨酸的突变体则没有。免疫共沉淀实验表明，野生型和带有FLAG标签的交换蛋白之间存在自缔合，而半胱氨酸-395突变为丙氨酸的突变不会抑制这种自缔合。这些结果表明，NCKX2以二聚体形式缔合，这种相互作用不需要通过半胱氨酸-395形成的二硫键，但可能会因该二硫键而稳定。这种连接可能通过限制蛋白质沿二聚体界面的移动性，从而降低NCKX2的转运活性。
