Figure 4. Activation of RyRs by photolysis of caged cADPR.

Sea urchin egg RyRs were reconstituted in lipid bilayers as described in Fig. 2, except that Ca²⁺ in the trans side was omitted and 1 mM EGTA and varying amounts of CaCl₂ were added to the cis side to set cytosolic [free Ca²⁺] to 10 μM (top traces in A) or 0.1 μM (second row of traces in A). A, in the presence of sea urchin egg homogenate, 10 μM [free Ca²⁺] in the cytosolic (cis) side elicited bursts of channel activity (first row of traces), which disappeared when cis [free Ca²⁺] was lowered to 0.1 μM (second row of traces). After a period of recording at [free Ca²⁺] = 0.1 μM, 7 μM caged cADPR was added to the cis side (third row of traces). No activity was detected during this period. A train of UV laser flashes was applied at 10 Hz frequency, shown by the horizontal dashed lines (bottom traces). The upward deflections of the baseline current are electrical artifacts created by the laser pulse. B, diary of activity of the RyR. Continuous records in control and after additions were divided into intervals of 30 s; P_o in each interval is plotted as a bar of length 0-1 with level 0 corresponding to no activity. The dotted line underneath the label UV flashes indicates the time in which laser UV flashes were applied at a frequency of 10 Hz. Three representative trains of channel activity elicited by UV pulses are shown (n = 6).