Depolarization-induced calcium uptake by vesicles in a highly enriched sarcolemma preparation from canine ventricle

Experiments were carried out to test the hypothesis that membrane depolarization stimulates Ca2+ uptake by vesicles in a sarcolemma preparation. Vesicles from a highly enriched sarcolemma preparation, previously loaded with 150 mM K+, developed a membrane potential when placed in a medium with 2.5 mM K+ as confirmed by changes in fluorescent intensity of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine. Inclusion of valinomycin in the assay increased the magnitude of the potential, and elevation of extra-vesicular K+, after development of the potential, caused depolarization. Ca2+ uptake by the vesicles was examined under three conditions: (a) nonpolarized state of the vesicles with 150 mM K+ on both sides of the membrane; (b) polarized state of the vesicles with inside negative due to high intravesicular K+ (150 mM) and low extravesicular K+ (2.5 mM); and (c) after a transition from a polarized to a relatively depolarized state by changing extravesicular K+ from 2.5 to about 106 mM. Ca2+ uptake was moderately affected by polarization of the vesicles after 10 min of reaction but was markedly and rapidly enhanced by depolarization of the vesicles. Extravesicular Na+ appeared to be required for the depolarization-induced Ca2+ uptake. La3+, Mn2+, and high concentrations of verapamil and tetrodotoxin inhibited the process. It was concluded that the effect of depolarization on Ca2+ uptake may reflect the process that serves as the trigger for myocardial contraction.