Thermodynamically irreversible gating of ryanodine receptors in situ revealed by stereotyped duration of release in Ca2+ sparks

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca2+ release underlying Ca2+ sparks in intact cardiac myocytes exhibits a prominent mode at similar to8 ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than similar to35 ms and a mode at similar to90 ms. These results indicate that, regardless of whether Ca2+ sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4 muM) and trans Ca2+ (10 mM), Suggesting that the irreversibility for Ca2+ spark genesis may reside at a supramolecular level. Modeling suggests that Ca2+-induced Ca2+ release among adjacent RyRs may couple the external energy derived from Ca2+ gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca2+ sparks.