A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2

被引:30
作者
Cai, XJ
Zhang, K
Lytton, J
机构
[1] Univ Calgary, Hlth Sci Ctr, Dept Biochem & Mol Biol, Cardiovasc Res Grp, Calgary, AB T2N 4N1, Canada
[2] Univ Calgary, Dept Physiol & Biophys, Calgary, AB T2N 4N1, Canada
关键词
D O I
10.1074/jbc.M208818200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In this study we have examined the roles of endogenous cysteine residues in the rat brain K+-dependent Na+/Ca+ 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.
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页码:48923 / 48930
页数:8
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