ATP‐Stimulated Ca²⁺ Transport into Cholinergic Torpedo Synaptic Vesicles

Abstract: Activation of the Ca²⁺/Mg²⁺ ATPase associated with highly purified Torpedo synaptic vesicles results in ⁴⁵Ca²⁺ uptake. The accumulated ⁴⁵Ca²⁺ is released by hypoosmotic buffer and by the Ca²⁺ ionophore A23187. Density‐gradient centrifugation and permeation chromatography reveal that vesicular acetylcholine and the membrane‐bound ⁴⁵Ca²⁺ co‐migrate, thus implying that ⁴⁵Ca²⁺ is transported into cholinergic vesicles. ATP‐dependent ⁴⁵Ca²⁺ uptake follows saturation kinetics, with K_mCa²⁺= 50 μM, K_mATP= 5 μM, and V_max= 3 ± 0.3 nmol Ca²⁺/mg protein/min. Treatment of the vesicles with mersalyl, dicyclohexyl‐carbodiimide, and quercetin leads to inactivation of the Ca²⁺/Mg²⁺ ATPase and to comparable inhibition of ⁴⁵Ca²⁺ transport. Ruthenium red and ouabain have no effect on either of these activities. Nigericin in the presence of external K⁺ is a potent inhibitor of ⁴⁵Ca²⁺ translocation, whereas gramicidin activates transport. The proton translocator carbonylcyanide p‐trifluoromethoxy‐phenylhydrazone (FCCP) and FCCP + the ionophore valinomycin partially inhibit ⁴⁵Ca²⁺ transport. By contrast, the above ionophores do not affect Ca²⁺/Mg²⁺ ATPase activity. Tentative mechanisms for ATP‐dependent Ca²⁺ transport into cholinergic synaptic vesicles and the physiological significance of this process are discussed.