Ryanodine sensitizes the Ca2+ release channel (ryanodine receptor) to Ca2+ activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca2+ signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca2+ release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca2+ dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg2+ and to activation by caffeine and ATP. In the presence of Mg2+, the ryanodine-modified single wt RyR2 channel displayed a Sigmoidal Ca2+ dependence with an EC50 value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC50 of 120 muM for Ca2+ activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca2+ activation by similar to1,000-fold. Furthermore, ryanodine is able to restore Ca2+ activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit similar to1,000-fold reduction in Ca2+ sensitivity to activation. The E3987A mutation, however, affects neither [H-3]ryanodine binding to nor the stimulatory and inhibitory effects of ryanodine on the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca2+.