Fast Release of Calcium from Sarcoplasmic Reticulum Vesicles Monitored by Chlortetracycline Fluorescence1

Abstract

Rapid Ca²⁺ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free. Ca²⁺ concentration. Ca²⁺ efflux was activated by extravesicular Ca²⁺ with an apparent dissociation constant of 25 μM and was inhibited with an inhibition constant of 120 μM in the absence of Mg²⁺. Caffeine enhanced the Ca²⁺ release rate by increasing only the affinity of Ca²⁺ for the activation site. Mg²⁺ reduced the Ca²⁺ release rate by competitive binding to the activation site. ATP increased the Ca²⁺ release rate very much without changing the affinities of Ca²⁺ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca²⁺ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca²⁺ release in the presence of ATP reached 80 s⁻¹. This value is considered to be sufficient to cause muscular contraction.