(Received for publication, September 18, 1995; and in revised form, October 20, 1995)
From the
The thiol-oxidizing reagent, thimerosal, has been shown to
increase the intracellular Ca concentration, to
induce Ca
spikes in several cell types, and to
increase the sensitivity of intracellular Ca
stores
to inositol 1,4,5-trisphosphate. Ryanodine-sensitive stores have also
been implicated in the generation of Ca
oscillations
induced by the addition of thimerosal. Here we report that micromolar
concentrations of thimerosal stimulate Ca
release
from skeletal muscle sarcoplasmic reticulum vesicles, inhibit high
affinity [
H]ryanodine binding, and modify the
channel activity of the reconstituted Ca
release
protein. Thimerosal inhibits ryanodine binding by decreasing the
binding capacity (B
) but does not affect the
binding affinity or the dissociation rate of bound ryanodine. Single
channel reconstitution experiments show that thimerosal (100-200
µM) stimulates single channel activity without modifying
channel conductance. The thimerosal-stimulated channel is not inhibited
by heparin. Furthermore, a Ca
-stimulated channel is
first activated and then inhibited in a time-dependent fashion by high
concentrations of thimerosal (1 mM). Once inactivated, the
channel cannot be reactivated by addition of either Ca
or ATP.
Several mechanisms have been proposed to explain oscillations in
the cytoplasmic Ca concentration observed during
fertilization of egg cells(1, 2, 3) .
Ca
released from inositol 1,4,5-triphosphate
(IP
) (
)and/or ryanodine-sensitive Ca
stores from the endoplasmic reticulum appears to be responsible
for these Ca
oscillations. It has been demonstrated
that micromolar concentrations of the sulfhydryl reagent thimerosal
(TMS) evoke similar repetitive Ca
spikes. The
IP
receptor has been strongly implicated in this process on
the basis of the observations that TMS stimulates Ca
release from liposomes containing the reconstituted purified
IP
receptor(4) , increases the affinity of IP
for receptor binding sites(5) , and increases the potency
of IP
-induced Ca
release (4, 6) . Moreover, a monoclonal antibody raised
against the IP
receptor blocks TMS enhanced
Ca
-induced Ca
release and
Ca
oscillations in hamster eggs(7) . In
contrast to these observations, the effects of ryanodine forced a
reevaluation of the role of IP
on the release of
Ca
from endoplasmic reticulum of oocytes. The
observation that the amplitude of Ca
oscillations in
response to sperm factor, IP
, or thimerosal decreased and
was eventually blocked by the addition of ryanodine suggested that
ryanodine-sensitive Ca
stores also play a role in
generating intracellular Ca
oscillations(3) .
The mechanism underlying thimerosal's effects on internal
endoplasmic reticulum Ca stores appears to involve an
interaction with sulfhydryl groups. The addition of reducing agents
such as dithiothreitol (DTT) has been shown to inhibit TMS potentiation
of IP
-induced Ca
release in sea urchin
eggs (9) and TMS-induced Ca
spikes in single
HeLa cells(6) . Furthermore, Ca
-sensitized
IP
-induced Ca
release has been shown to
be amplified by addition of oxidized glutathione
(GSSG)(5, 10, 11) . These results suggest
that the oxidative state of critical sulfhydryls on the IP
receptor (IP
R) modifies receptor activity and
responsiveness. It has been proposed that a highly conserved sequence
containing two cysteine residues located near the carboxyl terminus of
all subtypes of the IP
R and the ryanodine receptors are
likely targets for TMS regulation of channel activity(4) .
The Ca release mechanism from sarcoplasmic
reticulum (SR) is also sensitive to the oxidative state of critical
thiols found on the ryanodine receptor. Oxidation of sulfhydryl groups
to a disulfide results in 1) stimulation of Ca
release across SR vesicles, 2) contraction of skinned muscle fibers, 3)
modification of the gating characteristics of single Ca
channels reconstituted into a planar bilayer lipid membrane
(BLM), and 4) alteration in high affinity
[
H]ryanodine binding to its
receptor(12, 13, 14, 15, 16) .
Using a fluorescent coumarin maleimide (cpm) at nanomolar
concentrations, it has recently been demonstrated that hyperreactive
thiols on the RyR are disulfide-linked into a high molecular weight
complex during activation of Ca
release(17, 18) . This complex dissociates via
reduction upon closing of the Ca
release channel. If,
as recently proposed, conserved sequences found in both the
IP
R and the RyR are targets for TMS regulation of channel
activity(4) , then one would expect that the SR Ca
release mechanism would also be sensitive to TMS modification. In
this report, we demonstrate a direct interaction between TMS and the
Ca
release channel/ryanodine receptor from skeletal
muscle sarcoplasmic reticulum.
Ca uptake into SR vesicles (0.5
mg/ml) was carried out in a buffer containing 100 mM KCl, 20
mM HEPES, 1 mM MgCl
, 15 mM creatine phosphate, 5 units of creatine phosphokinase, 50
µM CaCl
, and 100 µM antipyrylazo
III. Uptake was initiated by the addition of 0.2 mM
Mg
ATP. Upon achieving steady state Ca
uptake, release was initiated by the addition of TMS, and the
free extravesicular Ca
concentration was recorded as
a function of time.
Following active accumulation of Ca, the
addition of micromolar concentrations of TMS induced Ca
release from SR vesicles (Fig. 1). Ca
release induced by TMS was not affected by the known channel
inhibitors ruthenium red (50 µM) and Mg
(10 mM) (not shown). When the reducing agent DTT (1
mM) was added following TMS-stimulated release of
Ca
(Fig. 1A), the effect was
partially reversed, and Ca
was reaccumulated by the
SR vesicles. Moreover, if TMS was added prior to activation of the
Ca
pump by ATP, Ca
uptake was
significantly diminished. Subsequent addition of DTT significantly
increased the amount of Ca
accumulated by these
vesicles (Fig. 1B). Furthermore, as the TMS
concentration was increased, the t
decreased
(corresponding to faster release rates), and the amount of released
Ca
saturates at nearly 100% (Fig. 1C).
Figure 1:
TMS-induced Ca release was inhibited by DTT. SR vesicles were incubated in 100
mM KCl, 20 mM HEPES, 1 mM MgCl
,
15 mM creatine phosphate (CP), 5 units of creatine
phosphokinase (CK), and 100 µM antipyrylazo III (AP III) at pH 7.0 to monitor extravesicular
Ca
. In A, after two additions of
Ca
(25 µM), ATP (0.2 mM) was
added to initiate Ca
uptake. The subsequent addition
of TMS elicited Ca
release. Addition of DTT (1
mM) reversed Ca
release induced by TMS and
resulted in the active reaccumulation of Ca
by the
SR. In B, TMS was added before ATP. Ca
uptake was significantly reduced. Subsequent addition of DTT
reversed the effect of TMS and promoted further uptake of
Ca
into the SR. In C, the half-time for
Ca
release (t
) and the
percent of the total releasable Ca
(%
Ca
release) were plotted as a function of the added
[TMS]. Abs., absorbance.
On the basis of the data presented in Fig. 1, release of Ca by TMS could be caused
by a specific interaction with the Ca
release
mechanism of the SR, by modification of the activity of the
Ca
pump(22) , by a nonspecific leakage, or by
a combination of these effects. A direct interaction with the
Ca
release protein/ryanodine receptor is demonstrated
in Fig. 2. As shown in Fig. 2A, TMS inhibited
high affinity ryanodine binding in a concentration-dependent manner,
with an IC
of
50 µM. A Scatchard plot (Fig. 2B) was fit to a one-site model using linear
regression analysis. This analysis indicated that inhibition of
ryanodine binding was caused by a decrease in the maximal number of
binding sites (B
decreased by
57% (2.54
pmol/mg for the control versus 1.09 pmol/mg in the presence of
50 µM TMS)), with no apparent modification in the
Ca
dependence of activation or inhibition of the
receptor (Fig. 2C). Moreover, the equilibrium
dissociation constant for ryanodine binding, K
,
was unmodified by TMS treatment (12.2 nM for the control and
10.4 nM with TMS) (Fig. 2B). The rate of
dissociation of bound ryanodine (k
) was also
unaffected by the presence of TMS in the dilution buffer (Fig. 2D), and it therefore can be concluded that the
association rate constant (k
) for ryanodine
binding is also independent of TMS concentration. In contrast to these
observations, Hilly et al.(5) has shown that TMS (100
µM) decreased the K
for IP
binding to permeabilized hepatocytes and cerebellar membranes
without affecting the maximal binding capacity (B
). Under similar conditions, Renard-Rooney et al.(8) observed a decreased K
for IP
binding with an increased receptor occupancy (B
).
Figure 2:
Thimerosal modified high affinity
[H]ryanodine binding to SR vesicles. SR vesicles
(0.1 mg/ml) were incubated at 37 °C for 3 h (A-C) in
a medium containing 250 mM NaCl, 15 mM KCl, 15
nM [
H]ryanodine, 20 mM HEPES,
pH 7.1. In A, B, and D, the assay buffer
contained 50 µM Ca
. In C, the
free Ca
concentration was calculated after the
addition of varying amounts of Ca
and EGTA. In B, 1 nM [
H]ryanodine and
varying concentrations of unlabeled ryanodine (0.5-64
nM) were incubated with SR vesicles in the presence or absence
of TMS (50 µM). In D, SR was incubated with 1
nM [
H]ryanodine for 3 h. Dissociation of
bound ryanodine was initiated by a 100-fold dilution into a binding
medium without ryanodine containing the indicated concentration of TMS.
Dissociation was quenched by rapid filtration at the indicated times.
In all assays, the binding reaction was quenched by rapid filtration
through Whatman GF/B glass fiber filters and rinsed twice with 5 ml of
buffer. The data shown are the average of representative experiments
performed in duplicate and repeated at least two
times.
Direct modification of the
Ca release mechanism was also demonstrated following
reconstitution of the release mechanism into a planar BLM. As shown in Fig. 3A, in the absence of added cis Ca
, 100-200 µM TMS stimulated
single channel activity. With a 5:1 CsCl gradient, the unitary
conductance (453 picosiemens) and selectivity
(P
/P
32) of the single channel activated by TMS (not shown) were the same as
previously reported(16) . The TMS-activated channel was not
affected by the IP
R inhibitor heparin (200-400
µg/ml) (not shown). Moreover, as in the case of vesicle flux
measurements (Fig. 1), activation of channel activity stimulated
by TMS (200 µM) was reversed by the addition of a reducing
agent (5 mM DTT) (Fig. 3B). Under conditions
of channel activation by TMS, treatment with DTT appeared to restore
the channel to its native configuration, and subsequent addition of 50
µM Ca
increased the channel open
probability (Fig. 3B, trace d). In contrast to
this behavior, in the presence of activating concentrations of
Ca
(50 µM), high concentrations of TMS
(1 mM) not only activated single channel activity but also
inhibited channel activity in a time-dependent manner (Fig. 3, C and D). Once inhibited by high concentrations of
TMS, single channel activity was not reactivated by subsequent addition
of 100 µM Ca
or 1 mM ATP (not
shown).
Figure 3:
Thimerosal modified the single channel
characteristics of the SR Ca release protein.
Following fusion of an SR vesicle to a planar BLM, single channel
current was recorded as a function of time. In the absence of added
Ca
(Ca
5
µM) (A, trace a), a control recording
was followed by two consecutive additions of 100 µM TMS (A, traces b and c). The channel open
probabilities for each trace are as follows: A (a), P
= 0.02; A (b), P
= 0.15; A (c), P
= 0.40. In B, DTT
reversed channel activation induced by TMS. Trace B (a) shows a control channel in the presence of 5
µM Ca
(P
= 0.01). Addition of 200 µM TMS (trace B (b)) activated channel activity (P
= 0.25), while 5 mM DTT (trace B (c)) decreased the activity of the
channel (P
= 0.05). Subsequent
addition of 50 µM Ca
(trace B (d)) reactivated channel activity (P
= 0.30). In the presence of 50 µM Ca
, high concentrations of TMS modified single
channel activity in a time-dependent manner (C). To a control
membrane (trace C (a) (P
= 0.03)), 50 µM Ca
was
added (trace C (b) (P
= 0.55)). One minute following the addition of 1
mM TMS (trace C (c)), the open probability
increased (P
= 0.95). Traces C (d) and C (e) illustrate inhibition of
single channel activity 80 and 100 s after the initial exposure to TMS (P
= 0.55 and P
= 0.10, respectively). In D, the time
dependence of the open probability for traces displayed in C are shown. At t = 0 s, 1 mM TMS was added
to the cis chamber, and the solution was stirred for 60 s. P
was measured in 5-s intervals. For all
traces shown (A--D), the holding potential was
+25 mV. The solid lines and arrows represent the
closed and open states of the channel, respectively. Each experiment
was repeated with similar results at least 4
times.
On the basis of vesicle flux, high affinity
[H]ryanodine binding, and single channel
measurements, it is clear that TMS directly interacts with the
Ca
release protein/ryanodine receptor from skeletal
muscle sarcoplasmic reticulum. TMS appears to be interacting with a
thiol group(s) associated with the Ca
release
mechanism of skeletal muscle
SR(12, 13, 14, 15, 16) .
Oxidation and reduction of critical sulfhydryl groups have been
postulated to be the mechanism underlying the gating of the
Ca
release
protein(13, 17, 18) . The oxidative state of
thiols present on both the RyR and the IP
R may be critical
in the regulation of channel gating. However, the changes in receptor
binding induced by TMS are somewhat different for the RyR and the
IP
R. Independent of the mechanism by which these channels
gate, TMS should not be used as a specific probe for interaction with
the IP
receptor. TMS has previously been shown to affect
the activity of the Ca
pump from rabbit skeletal
muscle SR and from rat cerebellar microsomes (22) and was shown
in this report to modify the gating characteristics and receptor
occupancy of the RyR/Ca
release protein from skeletal
muscle sarcoplasmic reticulum.