A disulfide bond is required for functional assembly of NCX1 from complementary fragments.
The cardiac Na(+)-Ca(2+) exchanger consists of a single polypeptide with two transmembrane segment (TMS) clusters separated by a large intracellular loop between TMS5 and TMS6 (Nicoll et al. (1999) J. Biol. Chem. 274, 910-917; Iwamoto et al. (1999) FEBS Lett. 446, 264-268). A "split" exchanger can be expressed by dividing the exchanger cDNA into two fragments so that the NH(2)- and CO(2)H-terminal portions of the protein are expressed as separate polypeptides in HEK293 cells. Expression of partial exchanger molecules did not result in detectable exchanger activity. Cells coexpressing both portions of the exchanger, however, displayed between 30 and 50% of the activity of the intact wild-type exchanger. The full-length exchanger contains a disulfide bond between residues 14 or 20 and 792. We examined the role of this disulfide bond in the split exchanger by mutagenesis and expression studies. Our results indicate that the function of the exchanger requires both TMS clusters and that the C(14 or 20)/C792 disulfide bond is essential for expression of active exchangers from half molecules.
Qiu
Z
Z
Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1760, USA.
Chen
J
J
Nicoll
D A
DA
Philipson
K D
KD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D004220:Disulfides; D010446:Peptide Fragments; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002118:Calcium