Figure 6. RyR dispersion during HF results in slowing of Ca²⁺ sparks.

To examine whether altered CRU morphology could slow Ca²⁺ spark kinetics in HF, spark profiles were simulated for a variety of dSTORM-derived RyR configurations. (A) Sparks were triggered by opening a single RyR (circled) which was randomly placed in consecutive simulations (example RyR opening trajectories are shown in the upper panels, with a family of spark time-courses illustrated below). Time to opening was registered for each RyR in the CRU, and the resultant time course of the Ca²⁺ spark was plotted until the final RyR closure, at which point the simulation was stopped for computational efficiency. Opening times were similar for individual RyRs within a solid, single cluster CRU, and the overall temporal profile of elicited sparks showed rapid kinetics which were rather consistent between consecutive simulations. By contrast, delayed and variable opening times were observed for individual RyRs in multi-cluster CRUs. This resulted in variable and slowed Ca²⁺ spark kinetics with these CRU configurations, as indicated by temporal spark profiles (A), a right-shifted distribution of time-to-peak measurements (B) and mean data (C). (*=P < 0.05 vs single-cluster CRU).

Figure 6—figure supplement 1. Effect of focal plane on Ca²⁺ spark detection and kinetics.

To examine whether out-of-focus Ca²⁺ release events could artefactually alter assessment of Ca²⁺ spark parameters, simulated Ca²⁺ release profiles were compared in ‘linescans’ collected at various depths of the 2 × 2 µm computational domain (54 RyR single cluster model). Vertical, stepwise displacement from the central plane resulted in a rapid decrease in measured spark amplitude (A). Indeed, events > 0.5 µm displaced from the focal plan were undetectable. While displacement of the focal plane also slowed apparent Ca²⁺ spark kinetics, such effects were negligible within the range where sparks were detectable (B).