BIOSYNTHESIS AND INITIAL PROCESSING OF THE CARDIAC SARCOLEMMAL NA-CA2+ EXCHANGER()

Based on the deduced amino-acid sequence of the cardiac Na+-Ca2+ excha nger, there are six potential N-linked glycosylation sites and a poten tial cleaved signal sequence. To study the post-translational modifica tions of the exchanger, in vitro translation was examined in the prese nce and absence of canine pancreatic microsomes. Glycosylation, detect ed as endoglycosidase H induced shifts in molecular size, was examined for proteins having different numbers of potential N-linked glycosyla tion sites by using full and partial length RNA transcripts. In the pr esence of microsomes, the molecular mass of the full-length clone incr eased from 110 to 113 kDa. Endoglycosidase H treatment led to a reduct ion to 108 kDa, indicating that glycosylation increases the molecular mass by approx. 5 kDa and a signal sequence of approx. 2 kDa is cleave d during processing. Analysis of molecular-mass shifts obtained with p artial 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 sar colemmal membrane or oocytes expressing the wild-type exchanger. The m olecular 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 u sed to determine the functional consequences of glycosylation. Na+-Ca2 + exchange current was examined in patches from oocytes expressing eit her 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 indica te that glycosylation does not affect exchanger function in Xenopus oo cytes and help to define exchanger topology.