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J. Biol. Chem., Vol. 277, Issue 50, 48923-48930, December 13, 2002
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From the Cardiovascular Research Group, Departments of Biochemistry
& Molecular Biology and Physiology & Biophysics, University of
Calgary, Calgary, Alberta T2N 4N1, Canada
In this study we have examined the roles of
endogenous cysteine residues in the rat brain
K+-dependent
Na+/Ca2+ 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
A Novel Topology and Redox Regulation of the Rat
Brain K+-dependent
Na+/Ca2+ Exchanger, NCKX2*
,
-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 -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.
*
This work was supported by grants from the Canadian
Institutes of Health Research and the Alberta Heritage Foundation for Medical Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported in part by a studentship from the Alberta Heritage
Foundation for Medical Research.
§
Senior Scholar of the Alberta Heritage Foundation for
Medical Research and an Investigator of the Canadian Institutes of
Health Research. To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, University of Calgary Health Sciences
Centre, Rm. 2518, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1,
Canada. Tel.: 403-220-2893; Fax: 403-283-4841; E-mail: jlytton@ucalgary.ca.
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