ELECTRICAL CURRENTS GENERATED BY A PARTIALLY PURIFIED NA CA EXCHANGERFROM LOBSTER MUSCLE RECONSTITUTED INTO LIPOSOMES AND ADSORBED ON BLACK LIPID-MEMBRANES - ACTIVATION BY PHOTOLYSIS OF CAGED CA2+/

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger, Immunoblots o f the lobster muscle and mammalian heart exchangers, following SDS-PAG E, indicate that the invertebrate and mammalian exchangers have simila r molecular weights: about 120 kDa, The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin v esicles which were loaded with 160 mM NaC1. Ca-45 uptake by these prot eoliposomes was promoted by replacing 160 mM NaC1 in the external medi um with 160 mM KC1 to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid memb ranes (BLM), and DM-nitrophen-Ca2+ (''caged Ca2+'') was added to the K C1 medium, photolytically-evoked Ca2+ concentration jumps elicited tra nsient electric currents. These currents corresponded to positive char ge exiting from the proteoliposomes, and were consistent with the Na/C a exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. Th e current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient, KC1-loaded proteol iposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations >25 mM reduced the c urrent, The dependence of the current on free Ca2+ was Michaelis-Mente n-like, with half-maximal activation (K-M(Ca)) at <10 mu M Ca2+. Caged Sr2+ and Ba2+, but not Mg2+ also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented t he Ca-dependent current, perhaps by facilitating the adsorption of pro teoliposomes to the BLM. The Ca-dependent current was irreversibly blo cked by La3+ (added as 200 mu M DMN-La3+). The results indicate that t he properties of the Na/Ca exchanger can be studied with these electro physiological methods.