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

General

A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2.
Authors:	Cai, X Zhang, K Lytton, J
Citation:	Cai X, etal., J Biol Chem 2002 Dec 13;277(50):48923-30.
RGD ID:	730066
Pubmed:	PMID:12377762 (View Abstract at PubMed)
DOI:	DOI:10.1074/jbc.M208818200 (Journal Full-text)
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.

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Biological Process

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Term	Qualifier	Evidence	With	Reference	Notes	Source	Original Reference(s)
protein-containing complex assembly		IMP		730066	dimerization	RGD

Molecular Function

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Term	Qualifier	Evidence	With	Reference	Notes	Source	Original Reference(s)
calcium, potassium:sodium antiporter activity		IMP		730066		RGD

Objects Annotated

Genes (Rattus norvegicus)

Slc24a2 (solute carrier family 24 member 2)

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