Figure 1. Force response of a skinned EDL fibre to 2 mm caffeine stimulation at different SR load levels.

The SR was completely emptied of its endogenous Ca²⁺ (left-most trace) by exposing the skinned fibre to the ‘full release’ solution (30 mm caffeine-low [Mg²⁺]-0.5 mm EGTA) (thick bar), with the area under the force response being indicative of the amount of Ca²⁺ released. The SR was reloaded to a different level by altering the load time (in a solution at pCa 6.7, 1 mm total EGTA; see Methods) before being fully depleted again. Exposure to 2 mm caffeine under the standard conditions (1 mm Mg²⁺; pCa 7.1, weakly Ca²⁺ buffered with 50 μm EGTA; see Methods) did not elicit any force response when the SR was loaded at approximately its endogenous level (17 s load), but did so if the SR was loaded to a considerably higher level (25 s load). (A small force artefact associated with moving the fibre out of one solution and into another can be seen immediately prior to the 2 mm caffeine exposure periods; a larger artefact often occurred when the fibre was moved into the air after the caffeine exposure.) Maximum force was determined by exposure to a 50 mm CaEGTA solution at pCa 4.5 (‘Max’). Time scale: 2 s during full release and 2 mm caffeine exposure, and 30 s during intervening wash/equilibration period and in ‘Max’.