A Thermal Transition of Passive Calcium Efflux in Fragmented Sarcoplasmic Reticulum

Abstract

The temperature dependence of passive Ca²+ efflux from skeletal muscle fragmented sarcoplasmic reticulum(FSR) was studied by dilution of a suspension of the vesicles into which 1 mM (CaCl₂ + ⁴⁵Ca) had been passively incorporated by overnight incubation at 3°. It was found that in the presence of 5 mM Mg²⁺, Ca²⁺ efflux could be resolved into two simultaneous first-order processes between 5° and 35°, but only a single first-order process appeared between 37° and 55°. Two independent functional transitions were found at 30°, indicating an abrupt membrane molecular reorganization at that temperature: (1) The two components of Ca²⁺ efflux at 5°-35° contributed equally to the total observed initial efflux at temperatures up to 30°. Between 30° and 35°, the relative contribution of the fast component progressively diminished until, by 37°, only the slow component remained. (2) The slow component, which persisted throughout the entire temperature range 5°-55°, exhibited a break in its Arrhenius plot at 30°-32°.

Elevation of internal Ca²⁺ concentration to 10 mM failed either to produce saturation kinetics of efflux or appreciably change its first-order rate constant. Omitting Mg²⁺ in the low temperature range accelerated Ca²⁺ efflux about 20-fold and eliminated the fast component, whereas including Ca²⁺ in the external medium in the high temperature range retarded Ca²⁺ efflux by about the same factor and generated a fast component. Omitting Mg²⁺ in the high-temperature range, however, had little effect on Ca²⁺ efflux. The failure of external divalent cation to stimulate Ca²⁺ efflux thus precludes an obligatory carrier-mediated exchange mechanism. Furthermore, participation of the catalytic turnover function of the Ca²⁺-ATPase molecule in Ca²⁺ efflux was unlikely because (1) the 30° transition temperature for efflux did not coincide with those previously determined for active Ca²⁺ uptake, ATPase activity, and reversal of the Ca²⁺ pump, and (2) above the transition temperature, the activation enthalpy and activation entropy increased for efflux but decreased for both active Ca²⁺ uptake and ATPase activity. Ca²⁺ efflux therefore probably involved simple diffusion through a membrane pore (Ca²⁺ “leak”). By comparison to the results of others using artificial and biological membranes, the effect of external divalent cation to produce a fast component of Ca²⁺ efflux from FSR is tentatively attributed to the formation of aggregates of SR vesicles.

Key Words:

• sarcoplasmic reticulum
• Ca²⁺ leak
• transport kinetics
• thermal transition
• permeability control
• vesicle aggregation