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J. Biol. Chem., Vol. 275, Issue 46, 35902-35907, November 17, 2000
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,,¶
From the Department of Molecular Biosciences, School
of Veterinary Medicine, University of California, Davis, California
95616 and the § Department of Anesthesia, Brigham and
Women's Hospital, Boston, Massachusetts 02114
Received for publication, August 4, 2000, and in revised form, September 19, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Inositol 1,4,5-trisphosphate receptors
(IP3R) and ryanodine receptors (RyR) mediate the release of
endoplasmic and sarcoplasmic reticulum (ER/SR) Ca2+
stores and regulate Ca2+ entry through
voltage-dependent or ligand-gated channels of the plasma
membrane. A prominent property of ER/SR Ca2+ channels is
exquisite sensitivity to sulfhydryl-modifying reagents. A plausible
role for sulfhydryl chemistry in physiologic regulation of
Ca2+ release channels and the fidelity of Ca2+
release from ER/SR is lacking. This study reveals the existence of a
transmembrane redox sensor within the RyR1 channel complex that confers
tight regulation of channel activity in response to changes in
transmembrane redox potential produced by cytoplasmic and luminal
glutathione. A transporter selective for glutathione is co-localized
with RyR1 within the SR membrane to maintain local redox potential
gradients consistent with redox regulation of ER/SR Ca2+
release. Hyperreactive sulfhydryls previously shown to reside within
the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical
component of a transmembrane redox sensor. Transmembrane redox sensing
may represent a fundamental mechanism by which ER/SR Ca2+
channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca2+ release from stores.
A change in cytosolic Ca2+ concentration serves as a
signal for modulating a wide range of cellular activities (1-3). A
major mechanism for increasing cytosolic Ca2+ includes
release of Ca2+ from internal stores (endoplasmic or
sarcoplasmic reticulum, ER or
SR)1 via a genetic superfamily of Ca2+ release
channels including inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR) (4-6). A prominent
functional property of all of these channels is exquisite sensitivity
to reduction and oxidation by sulfhydryl reagents (7-12). The
functional consequences of sulfhydryl modification of RyRs include
phases of activation and inhibition, revealing that multiple classes of
sulfhydryl groups residing on Cys residues of all three isoforms of RyR
channel complexes are important for native functioning and
subject to chemical modification (11, 12). However, defining a
role for sulfhydryl redox chemistry in RyR function has been controversial since the initial suggestion that sulfhydryl oxidation is
a key step in channel activation (13). A plausible physiological role
for redox control of ER/SR Ca2+ release channels and its
attendant mechanism has remained elusive.
It is known that glutathione (GSH) and glutathione disulfide (GSSG)
constitute the major redox buffer system of skeletal muscle and
many non-muscle cells (14, 15). In the typical mammalian cell, the
ratio of [GSH]/[GSSG] in the cytosol is To study redox regulation of RyR channel activity, the bilayer lipid
membrane (BLM) preparation affords precise control of the redox state
on both the cytoplasmic (cis) and luminal (trans) faces of the reconstituted channel by adjustment of the
[GSH]/[GSSG] ratio to form varied redox potentials. In the present
work, we provide direct evidence that RyR1 channel activity follows
transmembrane redox potential. Chemical labeling studies with CPM
indicate previously identified hyperreactive sulfhydryl moieties within
the RyR1 complex (18, 19) constitute an essential component of a unique
transmembrane redox sensor.
Preparation of SR Membranes--
Sarcoplasmic reticulum
membrane vesicles were prepared from back and hind limb skeletal
muscles of New Zealand White rabbits according to the method of Saito
et al. (20) with some modifications. During the SR
preparation, GSH and GSSG were included in the homogenization buffer,
and the glutathione RP was made to GSH and GSSG Stock Solutions--
GSH was dissolved in degassed
Hepes (20 mM) buffer, and the solution was adjusted to pH
7.0. Aliquots (~0.2 ml) were transferred to vials and sealed
after blowing with argon. The vials were stored at Transport Measurements by Light Scattering
Techniques--
Osmotically induced changes in microsomal vesicle size
and shape (21) were monitored at 400 nm at a right angle to the
incoming light beam, using a fluorimeter (F-2000, Hitachi). A decrease in light scattering reflected vesicular swelling, a consequence of
osmotic changes as GSH and GSSG transported into the vesicle lumen.
Briefly, SR vesicles (25 µg/ml) were equilibrated in a hypotonic
medium (5 mM K-PIPES, pH 7.0). The osmotically induced changes in light scattering were measured after the addition of a small
volume (<10% of the total incubation volume) of concentrated and
stock solutions of the compounds to be tested. No changes in light
scattering were observed when an identical assay solution was diluted
with the same volume of stock buffer lacking GSH or GSSH. Sucrose was
used to test the selectivity of GSH- and GSSG-induced changes in light
scattering because it was shown to be weakly permeable to SR membranes
(21). Flufenamic acid, a known anion transport inhibitor (17), was used
to test the specificity of GSH or GSSG permeation of junctional SR.
Preparation of GSH and GSSG Extracts from SR Lumen--
SR
vesicles (100 µg) were incubated in 1 ml of solution A (100 mM KCl, 20 mM MOPS, 100 µM
CaCl2), and an indicated combination of [GSH] and
[GSSG] at 37 °C for 15 min. The mixtures were then centrifuged at
16,000 × g for 25 min at 4 °C. Each supernatant was
carefully decanted from its respective pellet, and the latter was
rinsed three times with 1 ml of solution A. The pellets were resuspended and homogenized in 100 µl of buffer containing 1 N potassium Pi buffer, 0.5% CHAPS
(w/v), and alamethicin (22) (0.1 mg/mg protein) and then incubated for
30 min at 37 °C to permit the release of all vesicular glutathione.
Following incubation, 100 µl of 5% tricholoracetic acid in redox
quenching buffer (20 mM HCl, 5 mM
diethylenetriaminepentaacetic acid, 10 mM ascorbic acid) was added into the solubilization mixture and the extract centrifuged at 16,000 × g for 25 min. The supernatant
was collected, applied to a Microcon YM-3 (3000 molecular weight
cut-off, Millipore) and centrifuged at 16,000 × g for
1 h. The filtrate (~200 µl), which represented the total SR
luminal glutathione (GSH + GSSG), was analyzed for [GSH]/[GSSG] as
described below.
Measurement of GSH and GSSG Content--
The GSH and GSSG
content of each luminal extract was determined in a manner similar to
the method of Senft et al. (23), using the fluorescence
probe o-phthaldialdehyde (OPA) with a few modifications. To measure total glutathione (GSH + GSSG), 25 µl of
each extract was added into 65 µl of 1 N potassium
Pi buffer containing 100 µM dithionite to
fully reduce GSSG. The reaction mixture was incubated for 1 h at
room temperature, followed by addition of 80 µl of 0.1 N
potassium Pi. To quantify the total GSH product produced
from the reduction reaction, 20 µl of OPA (5 mg/ml) was introduced
into the mixture and permitted to incubate at room temperature for 90 min. OPA fluorescence was measured with a fluorescence
spectrophotometer (model F-2000, Hitachi) at excitation
365 nm/emission 430 nm. The portion of total glutathione attributable
to GSH was analyzed in the manner described for total glutathione
without the use of dithionite. To determine the original background
content of luminal SR GSH and GSSG before the addition of exogenous
glutathione, 100 µg of SR was incubated with alamethicin (0.1 mg/mg
SR protein) to release the glutathione to the solution bathing the SR
vesicles. These values were used as background correction for
determining the net exogenous GSH and GSSG transported into SR vesicles.
Single-channel Kinetics in BLM--
Reconstitution of RyR1 and
recording of channel activity were performed as previously reported
(24) with some modifications. RyR1 channels were reconstituted into
planar lipid bilayer (5:2 phosphatidylethanolamine:phosphatidylcholine,
Northern Lipids Inc., 50 mg/ml in decane) by introducing SR vesicles to
the cis chamber. The cis chamber contained 0.7 ml
of 250 or 500 mM CsCl, 50-200 µM
CaCl2,and 10 mM Hepes, pH 7.4, whereas the
trans side (virtually grounded) contained 50 or 100 mM CsCl, 50-100 µM CaCl2, and 10 mM Hepes, pH 7.4. Upon the fusion of SR vesicle into
bilayer, cis chamber was perfused, and the
cis/trans CsCl gradient were reversed. Single-channel
activity was measured using a patch clamp amplifier (Dagan 3900) at a
holding potential as indicted in each figure. The data were filtered at
1 kHz before being acquired at 10 kHz by a DigiData 1200 (Axon
Instruments, Foster City, CA). The data were analyzed using pClamp 6 (Axon) and CDA 1.0 (provided by Dr. G. Liu) without additional
filtering. The length of representative current traces was Junctional SR membranes from rabbit skeletal muscle prepared in
the presence of a [GSH]/[GSSG] redox buffer with a potential of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
30:1, thereby maintaining
very reduced redox potential (RP) of approximately 
220 mV (16). By
contrast, the RP of the ER lumen is significantly more oxidized
(approximately 180 mV) and is maintained with a 3:1 to 1:1 ratio of
[GSH]/[GSSG] (16, 17). Thus, the typical microsomal membrane within
which the RyR and IP3R reside is normally subject to
a large transmembrane RP difference of 40-50 mV with the lumen much
more oxidized than the cytosol (16, 17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
220 mV, which mimics the typical
cytoplasmic RP in vivo. The preparations were stored in 10% sucrose, 10 mM Hepes, pH 7.4, at 80 °C until needed.
20 °C for no
longer than 60 days. Once thawed and opened for use, the vial was
discarded. GSSG solution was also made and stored in a similar manner
except without degassing and argon protection. Samples of GSH and GSSG
were taken from both sides of the BLM chamber at the end of
channel recordings to verify that the initial redox potential did not
change during the course of the experiments. Redox buffers were found
to be stable for at least 1 h.
1 s.
Average Po was calculated from 1 min recording.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
220 mV (calculated from the Nernst relationship (16)) resulted in a
high percentage (43%) of reconstituted channels (n = 102) exhibiting a characteristic low open probability
(Po) gating mode (Fig.
1A). The remaining channels
(57%) exhibited a high Po gating mode. This
observation was consistent with those of previous reports (25-27).
Although the molecular details underlying low Po and
high Po gating modes has remained unclear, the
oxidation of one or more classes of sulfhydryl moieties was implicated
in stabilizing these experimentally observed gating modes. Low
Po and high Po gating modes
represent stable RyR1 channel gating behaviors that have been observed
in BLM experiments. These gating modes appeared to depend on the
overall redox state of the receptor complex because low
Po behavior was promoted by treatment with reducing agents, whereas high Po behavior was promoted by
oxidizing agents. A characteristic of these gating behaviors was that
once they were achieved, the channels did not require the continued presence of reducing or oxidizing equivalents to maintain their gating
modes. This observation strongly suggested that low
Po and high Po gating behavior
may result from the breaking or forming intra- of inter-subunit
disulfide bonds (25). This hypothesis was further supported by the
observation that gating behaviors can be interconverted by the
subsequent addition of reducing or oxidizing reagent. Although channels
exhibiting low and high Po gating modes were found
to be tightly responsive to transmembrane RP, the present study focuses
primarily on channels exhibiting the low Po gating
mode. To test the response of the RyR1 channel to cytosolic oxidation,
the channel was challenged with a highly oxidized RP of 180 mV
generated by adding [GSH]/[GSSG] at a ratio of 3:1 (total
[glutathione] = 4 mM) to the cis chamber of
the BLM (Fig. 1B). Surprisingly, the channel showed a
negligible change in gating activity (Po = 0.011).
However, immediately after the addition of 3:1 [GSH]/[GSSG] to
generate the same redox potential of 180 mV on the luminal
(trans) side of the channel, channel Po
increased 13-fold. (mean Po rose from 0.011 to 0.138 based on 2-4 continuous minutes of record before and after setting the
trans redox potential; Fig. 1C). If channel
activation were simply the result of inclusion of an oxidizing
potential of 180 mV on the luminal face of the channel, then the
higher channel activity should persist after removal cytoplasmic RP.
However, removal of the cytoplasmic RP by extensive perfusion of the
cis chamber with an identical solution lacking glutathione
resulted in a 13-fold decrease in channel Po (mean
Po from 0.138 to 0.011; Fig. 1D).
Responsiveness to transmembrane redox potential has been observed in 83 of 106 separate reconstitution experiments from junctional SR prepared with or without redox buffering in the initial steps (including channels exhibiting both low and high Po gating modes) and
appears to represent a common feature of RyR1 channel regulation.
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Fig. 1.
Transmembrane glutathione redox buffer is
essential for the transmembrane redox sensing of RyR1. A RyR1
channel from skeletal muscle SR vesicles was incorporated into BLM, as
described under "Experimental Procedures." The channel activity was
recorded in the presence of 7 µM free cis
Ca2+ at a holding potential of 40 mV (A). The
introduction of 3 mM GSH and 1 mM GSSG, which
corresponded to a RP of approximately 180 mV (16), into the
cis side had a negligible influence on channel activity
(B). Subsequent inclusion of the same [GSH]/[GSSG],
which gave 180 mV in the trans side, significantly
enhanced channel open probability (Po;
C). Immediately following the removal of GSH and GSSG from
the cis side by extensive perfusion of the cis
chamber, the channel Po returned to control levels
(D). Similar perfusion of the trans chamber to
remove the trans redox buffer did not further alter channel
activity (E). The figure shows the Po
histograms of 2-4 min of continuous record and an expansion of a
representative 2 s of current trace for each manipulation of redox
buffer. Mean Po was calculated from the entire 2-4
min of continuous data and denoted in each section of the figure. The
current fluctuation is downward; a dashed line is
the maximal amplitude; arrows labeled c and
o indicate closed and open levels,
respectively. This experiment was repeated for a total of 11 separate channels that gave similar results.
RyR1 channels responded to local changes in transmembrane RP
irrespective of the absolute concentration of glutathione used in the
buffer. Both the cytoplasmic and luminal sides of the membrane were set
to 180 mV using a variety of [GSH] and [GSSG] ranging from 0.1 to
4 mM (Fig. 2, top
panel, conditions a-d). RyR1 channels exhibited
Po values ranging from 0.014 to 0.084 in the presence of 10 µM cis Ca2+ and
undefined RP (no GSH/GSSG). RyR1 channels responded with a 7-8-fold
enhancement in gating activity once a symmetrical 180 mV
transmembrane RP was established (Fig. 2, lower panel). In n = 19 separate reconstitution experiments, channel
activity was enhanced to the same degree (7-8-fold of the respective
control activity in the absence of a defined transmembrane RP) and was independent of the absolute [GSH] and [GSSG] used to obtain a symmetrical 180 mV RP. These results suggest that RyR1 channel activity responds to changing transmembrane RP and may modify sensitivity to cytoplasmic Ca2+. Thus, one unique
physiologic role of redox sensing afforded to ER/SR Ca2+
channels is the continuous alignment of Ca2+-induced
Ca2+ release gain with small changes of transmembrane redox
potential. High concentrations of either GSH or GSSG (2
mM) added singly to the cytoplasmic side of the RyR1
channel resulted in persistent changes in channel gating behavior (data
not shown) as previously reported (28). These effects are likely due to
the extreme reduction and oxidation potentials that nonselectively
alter protein thiols/disulfides and adversely impact a physiologically
relevant transmembrane redox sensor (11, 12, 28).
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In healthy non-muscle cells, the ER transmembrane RP gradient is
maintained by one or more transporters that facilitate diffusion of
either GSH or GSSG and the process is blocked by flufenamic acid (17).
To establish the physiological relevance of a trans SR
membrane redox sensor within the RyR1 complex, we determined whether or
not junctional SR possesses a transport mechanism necessary to generate
a transmembrane redox gradient. First, light scattering measured
spectrally shows that transport of GSH or GSSG (10 mM) from
the extravesicular space into the vesicle lumen (Fig.
3A). The rate of decline in
fluorescence represents the uptake of GSH or GSSG into the SR vesicles
(thereby decreasing light scattering). Based on the initial rate,
junctional SR vesicles transported GSH ~5 times faster than GSSG,
consistent with recent measurements performed with liver microsomes
(17). Despite the higher initial rates of GSH over GSSG, the steady
state capacity of SR vesicles for GSH was only 2-fold higher than GSSG
within 10 min (
F = 40 versus 80; Fig.
3A). Transport of glutathione is selective because the
addition of equimolar sucrose to the transport medium produces only a
very small change in light scattering in the same time frame as
previously shown (21). Flufenamic acid is a known blocker of
glutathione transport across ER membrane vesicles prepared from liver
(17). Likewise, movement of GSH and GSSG across junctional SR was fully
inhibited by 1 mM flufenamic acid (Fig. 3A).
Second, we measured the transmembrane GSH and GSSG ratio at steady
state (10 min) using o-phthaldialdehyde as a quantitative
indicator (23). This method permits direct quantitative analysis of
total luminal glutathione (GSH + GSSG) and GSH. In this manner, the ratio of luminal GSH/GSSG can be measured in the presence of varying extravesicular RP Fig. 3B shows several ratios of
[GSH]/[GSSG] added to the SR buffer to give extravesicular RP
ranging from 231 mV to 180 mV. After a 10-min equilibration, the
vesicles were extracted and the relative concentration of GSH and GSSG inside the vesicle lumen determined using OPA as described under "Experimental Procedures." [GSH] is determined directly by adding OPA, whereas total [glutathione] ([GSH] + [GSSG]) was determined after reducing the sample with dithionite (23). Regardless of the
extravesicular redox potential set experimentally, the luminal ratio of
[GSH]/[GSSG] converged to 3:1 (Fig. 3B, fifth
column). These results reveal for the first time the ability of SR
lumen to clamp the ratio of its [GSH]/[GSSG] within narrow limits,
despite the presence varying cytoplasmic RP. A 3:1 ratio of
[GSH]/[GSSG] is consistent with a significantly more oxidized
microsomal lumen, as demonstrated previously (16, 17). Taken together,
the present results reveal that junctional SR membranes, such as ER
membranes from non-muscle origin (17), possess a selective transporter for GSH and GSSG. Although a common feature of this microsomal transporter is a preference for GSH over GSSG (based on initial rates),
steady state analysis reveals the ability of the ER/SR lumen to favor a
3:1 ratio of GSH/GSSG regardless of the cytosolic RP. A 3:1 GSH/GSSG is
consistent with the observation that healthy cells maintain an oxidized
luminal potential (~160 to 180 mV) relative to the cytosol
(~220 mV) (16). How the ER/SR lumen maintains an oxidized potential
despite the preference for transport of GSH is unclear. One possibility
is that GSH is oxidized to GSSG within the ER/SR lumen and that the
latter is preferentially retained (17). In support of this hypothesis,
there is evidence that GSSG can be formed locally within the ER lumen,
although the mechanism(s) remain obscure (17, 29, 30). The existence of
a GSH/GSSG transporter co-localized with RyR1 within junctional SR
membranes would be expected if transmembrane redox sensing is a
significant physiologic modulator of RyR1 function.
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The ability of the RyR1 complex to respond to transmembrane RP and the
apparent ability of SR/ER to create a transmembrane redox gradient
raises the possibility that RyR1 channel activity could be actively
regulated by subtle localized changes in transmembrane RP Fig.
4 show the typical response of RyR1
channels to small changes in transmembrane RP In the absence of a
defined RP, low Po channels exhibited infrequent
gating activity even when cis Ca2+ was present
in an optimal range (30-100 µM) Ca2+
(Fig. 4, A and C; top traces). Once
the transmembrane RP was buffered to 220/180 mV
(cis/trans), the Po increased 2.7-fold from 0.019 to 0.052 (Fig. 4A, middle trace).
However, upon adjusting cis to a more oxidized 180 mV, the
channel Po immediately increased an additional
2.1-fold (Fig. 4A, bottom trace) for a total enhancement of
nearly 6-fold. In this regard, RyR1 channels were under tight control
by transmembrane RP, regardless of their gating mode (low
versus high Po). Fig. 4B shows an example of a high Po channel closely following as little as +10-20 mV incremental jumps in RP on the cis side
relative to a fixed luminal RP of 180 mV.
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Modulation of channel Po is independent of the
direction in which the transmembrane RP is altered (reduction 
oxidation or oxidation reduction). For example, once the
transmembrane RP was set to symmetrical 180 mV, the
Po increased 4.2-fold compared with the initial
Po under undefined RP (Fig. 4C, compare
top and middle traces). Subsequent reduction of
cis to 210 mV reduced Po 2-fold. These
results reveal that RyR1 channels tightly follow the
cis/trans RP in both oxidizing and reducing directions.
Similarly, the Po of low Po RyR1
channels closely followed as little as 10 mV incremental jumps in RP
on the cis side relative to a fixed trans of
180 mV in transmembrane RP (Fig. 4D). In separate
experiments, the cytoplasmic RP was kept fixed at 160 mV and the
luminal RP incrementally titrated from 160 to 190 mV in 5 to 10
mV increments (n = 4, data not shown). As the RP
difference across the BLM increased, channel Po
increased accordingly (227% enhanced Po at 160 mV
cis versus 190 mV trans). These results reveal
that once RP is set on both the cis and trans
sides, RyR1 channel activity tightly follows incremental changes in RP
on either side of the membrane. Tight regulation of gating activity by
transmembrane RP may be likened to the tight voltage regulation
observed with voltage-gated ion channels, therefore representing a
fundamentally new mode in channel regulation.
The response of the RyR1 channels to a physiologically relevant redox
difference on cytoplasmic and luminal sides of the membrane indicates
that an essential component of the redox sensing mechanism may span the
ER/SR membrane and may involve the redox state of essential Cys
moieties. The redox difference across the ER/SR membrane may therefore
provide the driving force for electron transfer reactions that modify
the gating kinetics in response to physiological ligands. From what has
been known for many redox-sensitive biochemical processes, including
targets of transcription factors, antioxidants, cytokines, as
well as ion channels/transporters, cell growth-related genes, kinases,
phosphatases, etc., electron flow through CH2-SH moieties
of conserved Cys residues within proteins account for their
redox-sensing properties (14). An essential component of RyR1 redox
sensing may involve the transfer of electrons among two or more closely
spaced Cys moieties within the channel complex. The RyR1 complex has
been shown to possess a small number of highly reactive sulfhydryl
moieties in which chemical reactivity appears to dramatically increase
with decreasing Po by the presence of mM
Mg2+ and/or nM Ca2+ (18, 19, 24).
Consistent with previous reports (18, 19), the fast labeling kinetics
of these hyperreactive thiols by the fluorescent maleimide CPM
(7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin) took place
under conditions that favor RyR1 channel closure (e.g. 10 mM Mg2+). The hyperreactive SR thiols rapidly
formed thioether adducts with 0.2-1 pmol of CPM/µg of SR protein
(t1/2 of 11.67-15.37 s; n = 4, data
not shown). To determine whether chemical modification of hyperreactive
Cys moieties alter gating behavior, channels were arylated with CPM
under conditions known to promote selective labeling of only
hyperreactive Cys residues and the functional consequences determined
in the BLM. Fig. 5, A and
C, reveals that short-term (
5 min) exposure of a channel to 40 and 50 nM CPM, respectively, caused negligible
changes of Po (Fig. 5, A and
C) and mean open dwell time (Fig. 5B) (n = 8). Reconstitution of RyR1 channel from such
specifically CPM-relabeled SR vesicles also demonstrated unchanged
channel gating behavior (n = 13, data not shown).
However, prolonged incubation of CPM produced additional nonspecific
interactions with less reactive Cys residues and thus led to a
time-dependent irreversible inactivation of the channel
(Fig. 5C; n = 3). These results reveal that
formation of thioether adducts with the most reactive (hyperreactive) Cys residues within the RyR1 complex does not alter overt aspects of
channel gating behavior.
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Are the hyperreactive thiols associated with the RyR1 complex an
essential component of the transmembrane redox sensor? Fig. 6 addresses this important question by
comparing the responses of two separate channels to parallel changes in
transmembrane RP without and with chemical modification of the
hyperreactive Cys residues with CPM. Both channels responded strongly
to the instillation of a symmetric 180 mV transmembrane RP and were negatively regulated by a 220/180 mV cis/trans gradient
(compare Fig. 6, A and B, traces
2-4). The channel in panel B, after exposure to 20 nM CPM for 120 s, failed to respond to a 180/180
mV symmetric cis/trans redox gradient (Fig.
6B, compare traces 6 and 7), whereas the control channel maintained its redox-sensing properties (Fig. 6A, compare traces 6 and 7). Despite
the loss of transmembrane redox sensing, the CPM-modified channel, as
predicted, maintained unchanged gating behavior and sensitivity to 10 µM ryanodine (Fig. 6B, short trace,
labeled Ry). In separate experiments, RyR1 channels had been
reconstituted in BLM after hyperreactive thiols were specifically
arylated by CPM under the conditions described above. It was found that
these pretreated channels gated normally but lacked sensitivity to
transmembrane RP changes (n = 3, data not shown).
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Our findings reveal the existence of a transmembrane redox sensor
within a microsomal Ca2+ channel and represent the first
direct evidence linking transmembrane RP with a specific biochemical
mechanism regulating microsomal Ca2+ transport. Small
changes in localized RP appear to dramatically influence the RyR1
activity regardless of cytoplasmic [Ca2+]. Redox sensing
is therefore likely to have significant regulatory impact on
Ca2+-induced Ca2+ release. Considering the
broad distribution of IP3R and RyR receptors, redox control
of microsomal Ca2+ release channels may represent a
fundamental mechanism by which mammalian cells regulate
Ca2+ signaling and homeostasis in response to localized
changes in redox potential. Hyperreactive thiols within the channel
complex are an essential component of a transmembrane redox sensor,
which is likely to contribute important regulatory functions during normal intracellular signaling (mediated for example by nitric oxide) (31-34) and may be involved in mediating changes in
Ca2+ signaling during oxidative stress (24).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants 5RO1 AR43140 and 4PO1 AR17605.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular Biosciences, School of Veterinary Medicine, University of California, One Shield Ave., Davis, CA 95616. Tel.: 530-752-6696; Fax: 530-752-4698; E-mail: inpessah@ucdavis.edu.
Published, JBC Papers in Press, September 20, 2000, DOI 10.1074/jbc.C000523200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; BLM, bilayer lipid membrane; CPM, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin; GSH, glutathione; GSSG, glutathione disulfide; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; OPA, o-phthaldialdehyde; RP, redox potential; RyR, ryanodine receptor; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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