Molecular and mechanistic heterogeneity of the Na(+)-Ca2+ exchanger

1. Studying the effect of K+ on Na(+)-Ca2+ exchange in rat brain SPMs revealed that a consistent stimulation was obtained. This stimulation persisted also when FCCP was included in the K(+)-containing reaction mixture to minimize the effect of membrane potential on the electrogenic process. 2. Using Rb+ as a K+ analogue revealed that it was cotransported with Ca2+ in a Na+ gradient-dependent manner. The ratio between the amount of Ca2+/Rb+ transported in rat brain SPMs in a Na+ gradient-dependent manner suggests that not all the Na(+)-Ca2+ exchangers in that preparation cotransport Rb+ (K+) with Ca2+. This is supported also by the finding that Na+ gradient-dependent Ca2+ influx can proceed in rat brain SPMs in the complete absence of K+ although to a lesser extent. 3. Protein purification studies and immunological characterization indicate that a 70-kDa protein is consistently detected in rat brain SPMs. Immunological characterization of the proteins expressed in the 14-18 S mRNA-injected Xenopus oocyte in conjunction with Na+ gradient dependent Ca2+ uptake activity or in the same mRNA-fortified reticulocyte lysate suggest that proteins of about 70 kDa are specifically synthesized. 4. Torpedo electric organ Na(+)-Ca2+ exchanger differs at least in two respects from the rat brain Na(+)-Ca2+ exchanger: It has a low affinity to Na+ (K0.5 = 170 mM), and it reaches maximal activity between 15-20 degrees C. Reconstitution studies suggest that the temperature difference might reflect a difference in the proteins themselves rather then a difference in membrane fluidity due to a difference in the membrane lipid composition.