A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2.
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.
Cai
Xinjiang
X
Cardiovascular Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Alberta T2N 4N1, Canada.
Zhang
Kathy
K
Lytton
Jonathan
J
D000818:Animals; D001921:Brain / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*
D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D003545:Cysteine