Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement.
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.
Salvador
J M
JM
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.
Inesi
G
G
Rigaud
J L
JL
Mata
A M
AM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002846:Chromatography, Affinity; D006859:Hydrogen / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D011510:Proteolipids / Q000378:metabolism; D013552:Swine; D013574:Synaptosomes / Q000201:enzymology*
D002147:Calmodulin; D007476:Ionophores; D011510:Proteolipids; C020485:proteoliposomes; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D006859:Hydrogen; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium