A disulfide bond is required for functional assembly of NCX1 from complementary fragments

The cardiac Na+-Ca2+ exchanger consists of a single polypeptide with two tr ansmembrane segment (TMS) clusters separated by a large intracellular loop between TMS5 and TMS6 (Nicoll et aL (1999) J. Biol. Chem 274, 910-917; Iwam oto et aL (1999) FEBS Lett. 446, 264-268). A "split" exchanger can be expre ssed by dividing the exchanger cDNA into two fragments so that the NH2- and CO2H-terminal portions of the protein are expressed as separate polypeptid es in HEK293 cells. Expression of partial exchanger molecules did not resul t in detectable exchanger activity. Cells coexpressing both portions of the exchanger, however, displayed between 30 and 50% of the activity of the in tact wild-type exchanger. The full-length exchanger contains a disulfide bo nd between residues 14 or 20 and 792. We examined the role of this disulfid e bond in the split exchanger by mutagenesis and expression studies. Our re sults indicate that the function of the exchanger requires both TMS cluster s and that the C(14 or 20)/C792 disulfide bond is essential for expression of active exchangers from half molecules. (C) 2001 Academic Press.