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, while the ryanodine-unmodified single wt channel exhibited an EC50 of 120 µ M for Ca2+ activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca2+ activation by ~1,000 fold. Furthermore, ryanodine is able to restore Ca2+ activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit ~1,000 fold reduction in Ca2+ sensitivity to activation. The E3987A mutation, however, affects neither [3H]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 Ca 2+ . mutant RyR2 that exhibits a markedly reduced sensitivity to activation by Ca2+. Our results demonstrate that ryanodine-modified channels are sensitive to modulation, and that ryanodine increases dramatically the sensitivity of the RyR2 channel to activation by Ca2+.


INTRODUCTION
Ryanodine, a plant alkaloid, binds specifically with high affinity to and alters the function of intracellular Ca 2+ release channels (ryanodine receptors, RyRs). Because of its high affinity and specificity, ryanodine has been widely used as a specific ligand for the identification, purification, cloning, and functional characterization of RyRs (1)(2)(3)(4)(5)(6)(7). The unique and specific action of ryanodine on RyR function also has made it an invaluable pharmacological probe for intracellular Ca 2+ signaling in a variety of cells (8,9), and for understanding the mechanisms of ion conduction and channel gating of RyRs (10,11).
Binding of ryanodine to the high affinity site was correlated with channel activation, whereas occupation of the low affinity ryanodine binding site was related to channel inhibition (19)(20)(21). These opposite effects of ryanodine have been clearly demonstrated at the single channel level. Addition of micromolar concentrations of ryanodine caused the RyR channel to enter into a long-lived open state with a reduced single channel conductance (22,23). Exposure to high concentrations (submillimolar to millimolar) of ryanodine led to a persistent blockade of single RyR channels (11,21).
The molecular mechanisms underlying these complex changes in channel conductance and gating behavior of RyR upon ryanodine modification are not well understood.
[ 3 H]ryanodine binding studies have suggested that complete blockade of the RyR channel by high concentrations of ryanodine may result from the occlusion of the channel conduction pore by ryanodine (19). Detailed characterization of the effects of ryanodine or ryanodine derivatives on ion handling by RyR has been reported. These studies have led to the proposal that reduction in channel conductance upon ryanodine modification is likely to result from allosteric alterations in ion permeation and ion binding, and/or from partial block by ryanodine (10,24). Little is known about the molecular mechanism by which ryanodine increases the open probability (Po) of the RyR channels. Ryanodine is thought to 'lock' the RyR channel into a subconductance open state upon binding, and the ryanodine-modified RyR channel is thought to be insensitive to modulation by other ligands such as Ca 2+ , Mg 2+ , and ruthenium red (5,11,12,22).
The view that ryanodine 'locks' the RyR channel in an open substate has recently been contested by Tanna et al.(25) who observed that the Po of the channel before ryanodine modification influenced the Po of the ryanodine-modified state. Low Po channels displayed more closing events than high Po channels after modified by ryanodine. This observation suggests that ryanodine does not simply 'lock' the channel into an open state. This observation also implies that activation of the RyR channel by ryanodine would be finite and regulatable. To test these possibilities, we have examined the gating properties and the Ca 2+ dependence of the ryanodine-modified wild type cardiac ryanodine receptor (RyR2) and a 4 by guest on March 25, 2020 http://www.jbc.org/ Downloaded from mutant RyR2 that exhibits a markedly reduced sensitivity to activation by Ca 2+ . Our results demonstrate that ryanodine-modified channels are sensitive to modulation, and that ryanodine increases dramatically the sensitivity of the RyR2 channel to activation by Ca 2+ .

Materials
Ryanodine was obtained from Calbiochem. [

RESULTS
Ryanodine-modified single RyR2 channels were sensitive to modulation Early single channel studies showed that ryanodine increased the open probability (Po) of the RyR channel to near unity, and that at micromolar activating Ca 2+ concentrations, the ryanodine-modified channels were inhibited only partially by Mg 2+ and ruthenium red (22). These observations have led to the notion that ryanodine-modified RyR channels are insensitive to modulation (11). An alternative explanation for the partial inhibition is that at micromolar Ca 2+ concentrations, ryanodine activation may be too strong to be completely suppressed by Mg 2+ and ruthenium red. To test this possibility, we examined the effect of Mg 2+ on ryanodinemodified single RyR2 channels at nanomolar Ca 2+ concentrations. Fig. 1 shows that a single mouse wt RyR2 channel displayed brief openings and minimal Po at submicromolar Ca 2+ concentrations. Addition of ryanodine abruptly shifted the channel into a state with long-lived openings and a marked increase in Po, and a reduced single channel conductance (Figs. 1a,b).
The free Ca 2+ concentration was then reduced to ~ 12 nM by addition of 0.43 mM EGTA, which resulted in only a small reduction in Po (Fig. 1c). Subsequent addition of 2 mM MgCl 2 , however, decreased the Po of the ryanodine-modified channel markedly (Fig. 1d) Ryanodine increased the sensitivity of single RyR2 channels to activation by Ca 2+ To investigate the mechanism of ryanodine activation, we examined the Ca 2+ dependence of ryanodine-modified single wt RyR2 channels. A single RyR2 channel was modified by ryanodine in the presence of ~300 nM Ca 2+ and 2 mM Mg 2+ (Fig. 2Aa). The free Ca 2+ concentration was subsequently reduced to various levels by addition of EGTA. As seen in Fig Ryanodine restored the activity of the E3987A Ca 2+ sensing mutant To demonstrate further the sensitizing effect of ryanodine on Ca 2+ activation, we utilized a Ca 2+ sensing deficient mutant of RyR2. We have shown previously that a single point mutation E3987A reduced the sensitivity to Ca 2+ activation of single RyR2 channels by ~1,000 fold, as measured by single channel recordings in planar lipid bilayers (26). We reasoned that if ryanodine is able to enhance the sensitivity of the wt RyR2 channel to Ca 2+ activation by ~1,000 fold, ryanodine would be able to restore Ca 2+ activation of the E3987A mutant RyR2 channel to a level similar to that of the wt. To this end, we examined the Ca 2+ dependence of single E3987A mutant RyR2 channels after being modified by ryanodine. As shown in Fig. 4, upon modification by ryanodine, a single E3987A mutant RyR2 channel was converted to a state with a reduced single channel conductance and a markedly increased Po, as were observed with the wt channel ( Fig. 1) (26). It is important to note that the ryanodine-modified single Hence, ryanodine is able to restore Ca 2+ activation of the E3987A mutant RyR2 channels.
Furthermore, like that observed with the ryanodine-modified wt RyR2 channels, some ryanodine-modified E3987A mutant channels remained highly active even at ~100 nM Ca 2+ (Fig. 4e). These residual activities, however, could be abolished by subsequent addition of Mg 2+ (see below). These observations indicate that upon ryanodine modification the sensitivities to Ca 2+ activation of both the wt and E3987A mutant RyR2 channels were enhanced dramatically.
Ryanodine-restored E3987A Ca 2+ sensing mutant channels were sensitive to modulation by

Mg 2+ and caffeine
It is apparent from Fig. 4  It is of interest to know that treatment of wt RyR1 expressing cells with 40 mM caffeine plus 500 µM ryanodine resulted in a long-lasting Ca 2+ transient (35). If ryanodine at 500 µM were able to block completely the wt RyR1 channel, one would expect a short-lived Hence, the dramatically different outcomes observed with the wt and E3987A mutant RyR2expressing cells with or without ryanodine treatment are most likely to be the consequence of their marked differences in Ca 2+ sensitivity to activation, rather than the result of differential actions of ryanodine on the wt and E3987A mutant RyR2 channels.
In summary, we have demonstrated that ryanodine sensitizes the RyR channel to Ca 2+ activation and that ryanodine-modified channel is regulatable.    (26). The single channel shown was incorporated into the bilayer with its cytoplasmic side facing the cis chamber, based on its sensitivity to EGTA and Ca 2+ . The holding potential was +20 mV. Openings are upward.   RyR3. Traces shown are from a representative experiment that was repeated three times.
Similar results were observed. blocked within 120 seconds. The single channel shown was incorporated into the bilayer with its cytoplasmic side facing the cis chamber, based on its sensitivity to EGTA and Ca 2+ . All additions were made to the cis chamber. The holding potential was +20 mV. Openings are upward.