Solubilization and reconstitution of membranes containing the Na+ -Ca2+ exchange carrier from rat brain

The Na+ -Ca2+ exchange carrier from brain plasmalemma was solubilized in cholate and reconstituted into asolectin vesicles by the cholate dilution method. Optimal solubilization and reconstitution required the presence of high NaCl (greater than or equal to 1.3 M). The reconstituted vesicles rapidly accumulated 45Ca2+ in the presence of an outward directed Na+ gradient. Other monovalent ion gradients (K+, Li+ or cholate+) did not drive transport. Further, Mg2+ X ATP did not drive Ca2+ uptake in the reconstituted vesicles. Uptake was temperature dependent with highest uptake occurring at 37 degrees C. Intravesicular Ca2+ accumulated by the Na+ -dependent process could be released by the Ca2+ ionophore A23187 or by extravesicular Na+ but not by external EGTA. Ca2+ uptake was inhibited by extravesicular Li+ or Na+. The Ki for Na+ inhibition was 35 mM for both the original membrane vesicles from brain plasmalemma and for the reconstituted vesicles. Ca2+ uptake was saturable with respect to extravesicular Ca2+ (Km(Ca2+) = 27 microM).