SR Ca²⁺ content changes induced by long-lasting depolarizations of increasing amplitudes. (A) SR Ca²⁺ content changes monitored with Fluo-5N (upper traces) in response to 50-s depolarizing pulses of increasing amplitudes from a holding potential of −80 mV (lower traces) in the same fiber. Voltage pulses were given every 2 min. For each depolarizing pulse, the frequency of fluorescence image capture was 20 Hz until the end of the first second of the pulse, and then it was switched to 0.2 Hz to minimize photobleaching. The time interval between the two sampling frequencies is 5 s. Horizontal and vertical dotted lines indicate the initial level of fluorescence at −80 mV and the time at which the voltage pulse ended, respectively. (B) Relationship between the mean Fluo-5N fluorescence decay at the end of the voltage pulse (measured between the right arrowhead and the dotted line in the example trace) normalized to maximal Fluo-5N fluorescence change during the pulse (measured between the left arrowhead and the dotted line) and membrane potential (○) (n = 12). The relationship was fitted with a Boltzmann equation with V_1/2 of −18 mV and a slope factor of 18 mV. The relationship obtained in Fig. 1 B (•) was superimposed to allow comparison.