Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge...

The synaptosomal plasma membrane Ca2+-ATPase (PMCA) purified from pig brain was reconstituted with liposomes prepared by reverse phase evaporation at a lipid to protein ratio of 150/1 (w/w). ATP-dependent Ca2+ uptake and H+ ejection by the reconstituted proteoliposomes were demonstrated by following light absorption and fluorescence changes undergone by arsenazo III and 8-hydroxy-1,3, 6-pyrene trisulfonate, respectively. Ca2+ uptake was increased up to 2-3-fold by the H+ ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, consistent with relief of an inhibitory transmembrane pH gradient (i.e. lumenal alkalinization) generated by H+ countertransport. The stoichiometric ratio of Ca2+/H+ countertransport was 1.0/0.6, and the ATP/Ca2+ coupling stoichiometry was 1/1 at 25 degrees C. The electrogenic character of the Ca2+/H+ countertransport was demonstrated by measuring light absorption changes undergone by oxonol VI. It was shown that a 20 mV steady state potential (positive on the lumenal side) was formed as a consequence of net charge transfer associated with the 1/1 Ca2+/H+ countertransport. Calmodulin stimulated ATPase activity, Ca2+ uptake, and H+ ejection, demonstrating that these parameters are linked by the same mechanism of PMCA regulation.