Figure 2. Protocols.

Two protocols (normal and heavy Ca²⁺ loading) were applied to fill the SR with Ca²⁺. The normal loading protocol used 10 pulses from −55 mV to 0 mV for 200 ms at 1 Hz; whereas the heavy loading protocol used a two-step depolarization from −55 mV to 0 mV for 50 ms and then to 50 mV for an addition 150 ms in order to bring more Ca²⁺ in through I_NCX. Resting Ca²⁺ sparks were recorded for 15 s after either loading protocol. SR Ca²⁺ content was assessed by both the Ca²⁺ transient and the I_NCX induced by application of 10 mm caffeine ∼12 min after rupturing the patch. Example I_Ca, twitch and caffeine-induced Ca²⁺ transients, and I_NCX are shown.