A Ca2+-dependent Tryptic Cleavage Site and a Protein Kinase A Phosphorylation Site Are Present in the Ca2+ Regulatory Domain of Scallop Muscle Na+-Ca2+ Exchanger

Abstract Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na+-Ca2+exchanger. In the presence of 1 mm Ca2+, the major product was a peptide of ∼37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, ∼16- and ∼19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105–110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca2+. Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [γ-32P]ATP led to incorporation of 32P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca2+ regulatory domain of a scallop muscle Na+-Ca2+ exchanger that mediates direct modulation of secondary Ca2+ regulation by cAMP.