Biosynthesis and initial processing of the cardiac sarcolemmal Na(+)-Ca2+ exchanger

Based on the deduced amino-acid sequence of the cardiac Na(+)-Ca2+ exchanger, there are six potential N-linked glycosylation sites and a potential cleaved signal sequence. To study the post-translational modifications of the exchanger, in vitro translation was examined in the presence and absence of canine pancreatic microsomes. Glycosylation, detected as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosylation sites by using full and partial length RNA transcripts. In the presence of microsomes, the molecular mass of the full-length clone increased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduction to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleaved during processing. Analysis of molecular-mass shifts obtained with partial transcripts suggested that glycosylation occurs at position N-9. This was confirmed by site-directed mutagenesis studies. A molecular mass of approx. 120 kDa was measured for Western blots of cardiac sarcolemmal membrane or oocytes expressing the wild-type exchanger. The molecular mass was reduced by approx. 10 kDa for the N9Y mutant or from exchanger obtained from a baculovirus-infected insect cell line where glycosylation does not occur. The giant excised patch technique was used to determine the functional consequences of glycosylation. Na(+)-Ca2+ exchange current was examined in patches from oocytes expressing either the wild-type or N9Y mutant. The non-glycosylated mutant exhibited the same properties as the native exchanger with respect to voltage, sodium dependence, and the effects of chymotrypsin. The results indicate that glycosylation does not affect exchanger function in Xenopus oocytes and help to define exchanger topology.