Regulatory processes on the cytoplasmic surface of the Na+/Ca2+ exchanger from lobster exoskeletal muscle

A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger w as reconstituted with, presumably, random orientation in liposomes. Ca2+ ef flux from Ca-45-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na+ (Na-ev)-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+](ev)) with a half-maxima l activation at [Ca2+](ev) = 0.6 mu M. This suggests that the lobster muscl e exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most ot her species. In contrast, at low [Na+](ev), the Caev- binding site (i.e,, o n the cytoplasmic surface) for Ca transported via Ca2+/Ca2+ exchange was ha lf-maximally activated by about 7.5 mu M Ca2+. Mild proteolysis of the Na+/ Ca2+ exchanger by cr-chymotrypsin also upregulated the Na-ev-dependent Ca2 efflux. Following proteolytic digestion in Ca-free medium, the exchanger w as no longer regulated by nontransported ev Ca2+ Proteolytic digestion in t he presence of 1.9 mu M free ev Ca2+, however, induced only a 1.6-fold augm entation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medi um, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic d egradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by alpha-chymotrypsin. Additional ly, we observed a 6-fold increase in the Na+/Ca2+ exchange rate, on average , when the intra- and extravesicular salt concentrations were increased fro m 160 to 350 mM, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids .