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)Hryanodine 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$^2+$.
- 11507100
- acid,
- alanine,
- caffeine,
- calcium
- calcium,
- channel
- channel,
- dose-response
- drug,
- gating,
- glutamic
- gov't,
- humans,
- ion
- magnesium,
- mutation,
- non-u.s.
- receptor
- relationship,
- release
- research
- ryanodine
- ryanodine,
- support,
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