Figure 5.

Flux-dependent coordinated RYR2 channel activity. (A) Sample coordinated channel recordings (two channels present) and all-points histogram. The cytosolic solution contained 1 mM EGTA (0.45 µM of free Ca²⁺) and Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). Recordings (left) were made at 0 mV. Open events are upward, and zero current level is marked for the top recording by a thick solid line. The thin dashed lines (above zero current mark) indicate normal current level for one or two open channels. All-points histograms (right) were made from a long recording from which these sample recordings were taken. (B) Same channel and conditions as in A, but the 50 mM Ca²⁺ in the luminal solution was exchanged for Ba²⁺. (C) Same channel and conditions as in B, but the 50 mM Ba²⁺ in the luminal solution was changed back to Ca²⁺. (D) Ensemble traces were generated by aligning and summing many individual coordinated events (two channel events). Only the decay phase is shown here. Solutions are those described for A, except that the cytosolic free Ca²⁺ level was varied as indicated below. Trace marked “a” has a time constant of 47.49 ms and was generated from 2,308 events collected with 1 µM of cytosolic free Ca²⁺ present. Trace marked “b” has a time constant of 33.65 ms and was generated from 1,252 events collected with 0.7 µM of cytosolic free Ca²⁺ present. Trace marked “c” has a time constant of 18.62 ms and was generated from 792 events collected with 0.45 µM of cytosolic free Ca²⁺ present. The inset plots attrition time constants (ms) as a function of cytosolic free Ca²⁺ concentration (µM). Open circles are the time constants determined by single-exponential fits to the ensemble traces. The connected filled points are the predicted stochastic attrition calculated using Eq. 1, assuming two channels are present. This calculation assumes the single-channel Po’s as shown in Fig. 1 B and single-channel MOTs as shown in Fig. 3 D (inset). For example, single-channel Po’s were 0.03, 0.09, and 0.19 for 0.45, 0.7, and 1 µM of cytosolic free Ca²⁺, respectively. Single-channel MOTs for these same Ca²⁺ levels were 1.04, 6.86, and 11.46 ms, respectively.