Abstract
Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) 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 Ca(2+) 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 Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]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+).
Highlights
Ryanodine, a plant alkaloid, binds with high affinity to and alters the function of intracellular Ca2ϩ release channels.1 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 molecular mechanisms underlying these complex changes in channel conductance and gating behavior of RyR upon ryanodine modification are not well understood. [3H]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]
It is generally believed that, upon binding, ryanodine locks the RyR channel into an open state, and that the ryanodinemodified RyR is insensitive to modulation [5, 11]
Summary
RyR, ryanodine receptor; wt, wild type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; KRH, Krebs-Ringer-Hepes. 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]. Low Po channels displayed more closing events than high Po channels after modified by ryanodine This observation suggests that ryanodine does not lock the channel into an open state. This observation 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 Ca2ϩ dependence of the ryanodine-modified wild type cardiac ryanodine receptor (RyR2) and a mutant RyR2 that exhibits a. 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 Ca2ϩ
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