(Received for publication, August 13, 1996, and in revised form, November 11, 1996)
From the Departments of Molecular Physiology and
Biophysics and § Medicine, Baylor College of Medicine,
Houston, Texas 77030
Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.
The Ca2+ release channel of skeletal muscle
sarcoplasmic reticulum is a homotetramer with a subunit molecular mass
of 565 kDa (1). The channel opens in response to a signal from the
transverse tubules, which is triggered by membrane depolarization (2), and the resulting flux of Ca2+ from the sarcoplasmic
reticulum initiates the sequence of events that leads to muscle
contraction. Several laboratories have clearly demonstrated that the
activity of the Ca2+ release channel is modulated by
oxidation-reduction reactions (3-11). Oxidizing agents that stimulate
Ca2+ release from the sarcoplasmic reticulum include
H2O2 (3), 2,2-dithiodipyridine,
4,4
-dithiodipyridine (4), Cu2+ phthalocyanine dyes (5),
anthraquinone doxorubicin (6, 7), and thimerosal (8). Heavy metals such
as Hg2+, Ag+, Cu2+,
Cd2+, and Zn2+ have also been reported to
induce Ca2+ release, either by directly interacting with a
sulfhydryl or by causing oxidation (9). These studies led Abramson and
Salama (10) to propose a model for redox modulation of the channel that
involves three different sulfhydryl groups that exist in close
proximity and that can form mixed disulfides to open or close the
channel. The evidence that the channel can be altered by oxidation is
conclusive (3-11); the question is whether this oxidation plays a
physiological role in skeletal muscle. It is not yet known if disulfide
interchange or oxidation-reduction of sulfhydryls on the
Ca2+ release channel contribute to normal
excitation-contraction coupling, but an increasing body of evidence
suggests that such a mechanism could be an important modulatory
element. Under basal conditions, unfatigued skeletal muscle produces
reactive oxygen species (12) and nitric oxide (NO) derivatives (13, 14)
that have been shown to modulate excitation-contraction coupling (12,
13). Strenuous contractile activity increases reactive oxidant
production (14-17), which contributes to fatigue of both isolated
muscle preparations (15, 16, 18, 19) and human muscle in
vivo (20). Redox modulation of the ryanodine-binding protein has
been proposed as a common mechanism for these effects (21).
Proteins other than the Ca2+ release channel may contribute to its modulation by oxidizing agents. Pessah and co-workers (22, 23) have suggested that oxidation may involve the cross-linking of triadin to the Ca2+ release channel. The functional role of triadin in skeletal muscle and its relationship to the Ca2+ release channel have remained elusive. Caswell and co-workers (24) have suggested that it is involved in coupling of the Ca2+ release channel to the t-tubule voltage sensor. Others (25) on the basis of its putative arrangement in the membrane have argued against a role for triadin in connecting the voltage sensor to the Ca2+ release channel. There is, however, general agreement that this protein interacts with the Ca2+ release channel (24, 26). The functional significance of this interaction is not yet known.
In addition to modulation by oxidation-reduction, the Ca2+ release channel is also sensitive to reagents that react with free sulfhydryls but do not form disulfide bonds. N-Ethylmaleimide (NEM),1 a sulfhydryl alkylating agent, activates Ca2+ release at low concentrations, while it inhibits release at higher concentrations (27).2 NEM, however, induces Ca2+ release with a slower onset than the heavy metals, a finding that has been interpreted to mean that the sulfhydryls responsible for the observed effects on Ca2+ release are in a hydrophilic environment (9). Quinn and Ehrlich (28) reported that modification of cysteines on the Ca2+ release channel by methiosulfonate compounds reduces the conductance of the channel. The reaction occurs only when the channel is in the open state. These compounds reduce the conductance in multiple steps until complete closure of the channel is obtained, suggesting a reactive sulfhydryl in the ion conducting pathway. The multiple effects of sulfhydryl reagents on the activity of the Ca2+ release channel raise the question of whether all of these reagents are reacting with the same sulfhydryls or whether alteration of multiple classes of sulfhydryls can alter the function of the channel.
Each subunit of the Ca2+ release channel tetramer has 100 cysteines (1). In this report we attempt to distinguish between classes of functionally important sulfhydryls. We show that the alkylating reagent NEM has three distinct functional effects on the channel and the oxidizing agent, diamide (29), produces intersubunit cross-links within the tetrameric channel. Additional information about the location of the cross-links is obtained using a membrane preparation that has both the full-length 565-kDa subunit and the 400-kDa fragment generated by the action of endogenous calpain.
[3H]Ryanodine (61.5 Ci/mmol) was purchased from DuPont NEN. Ryanodine was obtained from Calbiochem. Diamide (15), NEM, and dithiothreitol (DTT) were obtained from Sigma. Phosphatidylethanolamine (bovine heart) and phosphatidylcholine (bovine brain) were obtained from Avanti Polar Lipids, Inc (Alabaster, AL). Ultima Gold scintillant was purchased from Packard (Meriden, CT).
Sarcoplasmic Reticulum (SR) Membrane PreparationSR membranes were prepared from rabbit backstrap and hindleg skeletal muscle and were purified by using sucrose gradient centrifugation as described elsewhere (30, 31). Protein was estimated by the method of Lowry et al. (32), using BSA as standard.
Cross-linkingFor binding experiments, membranes in buffer I (300 mM NaCl, 100 µM CaCl2, 50 mM MOPS (pH7.4)) were incubated with 100 to 500 µM diamide for 30 min at 4 °C or for 10 min at room temperature. Diamide was removed either by dilution (10-fold) or by pelleting and washing the membranes in a Beckman Airfuge by centrifuging 4 min at 30 p.s.i. For bilayer experiments the sample was incubated for 10 min at room temperature with 500 µM diamide in buffer II (225 mM CsSO3CH3, 10 µM CaCl2, 10 mM MOPS, pH 7.4). Membranes cross-linked in buffer I or II were washed and assayed for [3H]ryanodine binding and for changes in gel patterns by SDS-PAGE. All [3H]ryanodine binding assays were done in buffer I.
SDS-PAGE Electrophoresis in One and Two DimensionsSR membranes (20-40 µg) were cross-linked with diamide for 10 min at room temperature (23 °C) or 30 min on ice. The samples were treated with 5 mM NEM for 20 min at room temperature before solubilization in sample buffer. Electrophoresis on 5% SDS-PAGE was performed for the first dimension. Electrophoresis was continued for 15 min after the dye front ran off gel. The lanes were excised and used for second dimension electrophoresis. The excised gel strips were treated with 50 mM DTT in sample buffer without SDS and bromphenol blue for 60 min at room temperature (23 °C). The gel strips were loaded on the second dimension gel (5% SDS-PAGE). The gap was sealed with melted agarose (1% agarose, 2% SDS, 50 mM DTT). After electrophoresis, the second dimension gel was silver-stained.
Labeling Membranes with [14C]NEM and Quantitation of Incorporation into the 565-kDa BandMembranes (6 mg/ml) were
incubated with 1 mM [14C]NEM (lot 3120256, 40 mCi/mmol, 2.5 mM) for the times indicated in Fig. 9. The
reaction was stopped by the addition of 20 mM DTT and the samples were electrophoresed on 5% SDS gels. After staining with Coomassie Brilliant Blue, the 565-kDa band was excised, dried, and
rehydrated in 100 µl of H2O, digested with 7.5% TS-2
tissue solubilizer (Beckman) in 4 ml of nonaqueous scintillant (2.8 g/liter diphenyloxazole, 0.28 g/liter
p-bis-[2-(5-phenyloxazolyl)]benzene in toluene). Samples
were shaken for 8 h and counted after 48 h.
Equilibrium [3H]Ryanodine Binding
[3H]Ryanodine was incubated overnight (15-17 h) at room temperature (23 °C) with 5-75 µg of SR membranes in 100-250 µl of buffer containing 0.3 M NaCl, 50 mM MOPS (pH 7.40), 100 µg/ml BSA, 0.1% CHAPS, 100 µM CaCl2, 100 µM phenylmethysulfonyl fluoride, 200 µM aminobenzamidine, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin A. Nonspecific binding was defined in the presence of either 10 µM or 100 µM ryanodine. Bound [3H]ryanodine was separated from free by rapid filtration of the sample through Whatman GF/F glass fiber filters followed by five 3-ml washes with ice-cold wash buffer containing 0.3 M NaCl, 100 µM CaCl2, and 10 mM MOPS (pH 7.4). The radioactivity bound to the filters was quantitated by liquid scintillation counting of the filters in 5 ml of Ultima Gold scintillant.
Bilayer TechniquesPlanar bilayers consisting of 8:2
L--phosphatidylethanolamine and
L-
-phosphatidylcholine were formed following the
Mueller-Rudin procedure across a 100-µm diameter aperture in Teflon
cups as described previously (34). The mixture of phospholipids was dissolved in n-decane (Sigma) at a
concentration of 50 mg/ml. Both chambers were filled with buffer
solution (20 mM CsSO3CH3, 10 µM CaCl2, 10 mM MOPS, pH 7.4).
After bilayer formation, 5 µl of SR membranes were added to the
cis chamber to give a final protein concentration of 0.5 µg/ml. The other side of the bilayer was defined as trans
and is the ground. An osmotic gradient was formed between the
cis and trans chambers by adding a concentrated salt solution (4 M CsSO3CH3, 10 µM CaCl2, 10 mM MOPS, pH 7.4) to
the cis chamber. Recording solutions contained 225 mM CsSO3CH3 in both the
cis and trans chambers. All additions were made
to the cis chamber. Agar/KCl bridges were used to connect
the chambers to Ag/AgCl electrodes immersed in 2 M KCl. The
holding potential was +40 mV. The data were filtered at 2.5 KHz and
digitized at 10 KHz.
[3H]Ryanodine binding data were analyzed with nonlinear curve fitting using SigmaPlot (Jandel Scientific). Nonspecific binding was subtracted prior to analysis.
Single-channel recordings were analyzed using FETCHAN and pSTAT software (Axon Instruments, Inc). Steady-state open probabilities (Po) were determined by measuring the fraction of time that the channel is open in at least 2 min of recording. Instantaneous open probabilities were determined as the fraction that the channel was open in 10 s of recording.
NEM is an alkylating reagent,
which reacts primarily with cysteine residues (35). The effect of NEM
alkylation on the activity of the Ca2+ release channel
reconstituted into planar lipid bilayers is shown in Fig.
1A. Immediately after the addition of NEM,
the channel was inhibited (phase 1). Continued exposure to the
alkylating agent (3-10 min) produced a substantial activation (phase
2), but longer incubations led again to inhibition of channel activity (phase 3). This stepwise effect of NEM is shown in Fig. 1B
in the plot of Po values as a function of time
after the addition of 5 mM NEM. To investigate the phase 1 inhibition, we examined the effect of 200 µM NEM on the
activity of the channel in the bilayer (Fig. 1C). Only the
phase 1 inhibition was seen with 200 µM NEM, but the
subsequent addition of 5 mM NEM led to channel activation.
These effects of stepwise addition of NEM on Po
values are summarized in Fig. 1D.
NEM also has time-dependent effects on
[3H]ryanodine binding. The effect of reaction of the
membranes with 5 mM NEM is shown in Fig.
2A. For binding experiments, NEM was reacted
in binding buffer at 4 °C and the alkylation was stopped by the
addition of 10 mM DTT. Similar to the effects on channel
activity, there were three distinct phases of the alkylation that
altered [3H]ryanodine binding. The three phases are an
initial and rapid inhibition (phase 1), a recovery or enhancement of
binding (phase 2), and a second inhibitory step (phase 3). The phase 1 decrease with 5 mM NEM reaches 49.1 ± 4.4%
(n = 3) inhibition prior to the onset of phase 2. The
time course of these three phases was dependent on the concentration of
NEM (Fig. 2B). Lower concentrations of NEM (500 µM) slow the onset of phases 2 and 3, allowing phase 1 to
plateau at 54.6 ± 5.7% (n = 4) inhibition. If
the sulfhydryls that have reacted at 500 µM NEM are
primarily phase 1 sulfhydryls, it should be possible to add 5 mM NEM to membranes at the plateau stage and recover the
phase 2 enhancement of [3H]ryanodine binding. This
experiment is shown in Fig. 2C. The addition of 5 mM NEM 25 min after the 500 µM NEM addition
gave rise to the recovery of binding (phase 2) followed by phase 3 inhibition. The reaction with NEM under bilayer conditions was faster
than that obtained using the protocol employed for Fig. 2
(A-C). For comparison, the effect on
[3H]ryanodine binding of the reaction of membranes with
500 µM and 5 mM NEM in bilayer buffer and at
room temperature is shown in Fig. 2D. The three phases were
still seen, and the time of onset of each phase corresponded to those
observed in the bilayer; however, the extent of inhibition was always
less under bilayer conditions, possibly reflecting the difference in
the redox state of the channel under these conditions. All membrane
preparations that we tested showed the three-phase effect of NEM. Some
variation in the magnitude of the three phases was, however, observed.
Some air oxidation of sulfhydryls did occur, and the difference in the
three phases may reflect differences in the oxidation state of the
membranes.
Diamide Treatment Increases Open Probability of the Channel, Enhances [3H]Ryanodine Binding, and Forms Intersubunit Cross-links
The sulfhydryl oxidizing agent, diamide, can also
activate the channel reconstituted into planar lipid bilayers (Fig.
3). This activation was reversed by the addition of 5 mM DTT. The Po of the channel was
increased from 0.035 ± 0.007 (n = 3) to 0.132 ± 0.030 (n = 3) in the presence of 250 µM diamide. Subsequent treatment with DTT decreases the
Po to 0.022 ± 0.006 (n = 3).
Treatment of membranes with diamide caused an alteration in the
electrophoretic mobility of the 565-kDa subunit of the Ca2+
release channel. In diamide cross-linking experiments with either membranes (Fig. 4A) or purified
Ca2+ release channel,3 the
565-kDa band disappeared and a new band was seen with an electrophoretic mobility consistent with dimer formation. Approximate molecular mass values of the oligomers were determined using a 200-kDa
myosin standard and the full-length ryanodine-binding protein as a
565-kDa standard. Higher concentrations of diamide produced higher
oligomers of the 565-kDa standard, which did not enter the gel. To
demonstrate that the high molecular weight bands generated by diamide
treatment are indeed dimers formed by intersubunit cross-linking and
not by the cross-linking of the Ca2+ release channel to
other proteins, we performed two-dimensional electrophoresis, reducing
the disulfides between the first and second dimensions (37). The
two-dimensional SDS-PAGE is shown in Fig. 4B. The membranes
used in the experiment shown in Fig. 4B had a significant
amount of a 400-kDa fragment of the Ca2+ release channel,
which is derived from the 565-kDa band by calpain digestion (38, 39).
Both bands underwent cross-linking to form higher molecular weight
complexes. Upon reduction prior to the second dimension (Fig.
4B), the high molecular weight bands decreased to 565-kDa
and 400-kDa bands. Surprisingly, very little cross-linking was detected
between the 400-kDa and the 565-kDa proteins. Instead both the 565-kDa
and the 400-kDa proteins appeared to form only homodimers, suggesting
that, when the 565-kDa is digested to the 400-kDa, the other subunits
within the tetramer are also digested. Although only high molecular
weight bands are shown in this gel, we have extensively searched for
lower molecular weight bands such as triadin and have found no evidence
that any other protein is involved in the diamide-induced cross-linking to the Ca2+ release channel (data not shown).
To determine if the formation of intersubunit cross-links correlates
with channel activation, membranes were treated with increasing
concentrations of diamide. The effect of these treatments on
[3H]ryanodine binding is shown in Fig.
5A. Diamide treatment enhanced [3H]ryanodine binding. To quantitate the formation of
dimers, the Coomassie Brilliant Blue-stained gel was scanned with a
densitometer and the optical densities of the dimer and monomer were
plotted as a function of diamide concentration (Fig. 5B).
The disappearance of the 565-kDa band correlated with the appearance of
the dimer and with enhanced [3H]ryanodine binding. At
higher concentrations of diamide (1 mM and higher), the
intensity of the dimer also decreased and higher oligomers appeared to
accumulate at the top of the gel. Formation of higher oligomers was
accompanied by a decrease in [3H]ryanodine binding.
Channels pretreated with diamide and washed prior to incorporation into
planar lipid bilayers showed a substantial activation compared to
controls (Fig. 5C). In the experiment shown, the
Po increased from 0.02 to 0.22 with 500 µM diamide.
Diamide and NEM Can Be Used to Differentiate between Classes of Sulfhydryls on the Ca2+ Release Channel
To determine
whether diamide and NEM react with the same sulfhydryls, we examined
the effect of NEM on the diamide-activated channel. The single-channel
records are shown in Fig. 6. The first tracing is the
control (Po = 0.03 ± 0.01, n = 3). Addition of 250 µM diamide
activated the channel (Po = 0.12 ± 0.03, n = 3). The addition of 5 mM NEM to the
diamide-modified channel stimulated channel activity further. The
Po with NEM was 0.53 ± 0.04 (n = 3) within the first 5 min. This activation is not
reversed by DTT (data not shown). NEM does not cause phase 1 inhibition
in the diamide-pretreated membranes. Sulfhydryls involved in channel activation by diamide, therefore, appear to be different than those
involved in activation by NEM. Continued incubation with NEM leads to
channel inhibition. Additional activation by NEM is also seen with
channels pretreated with higher concentrations (500 µM to
1 mM) of diamide (data not shown).
To examine further the relationship between sulfhydryls altered by
diamide and those that react with NEM, we examined the effect of
pretreatment of membranes with diamide on the ability of NEM to alter
[3H]ryanodine binding (Fig.
7A). In these experiments the membranes were
reacted first with 250 µM diamide for 30 min and then
with 5 mM NEM. At various times after NEM addition,
aliquots were removed and the reaction stopped by the addition of DTT.
The samples were then assayed for [3H]ryanodine binding.
As can be seen in Fig. 7A, diamide pretreatment prevented
the phase 1 inhibition of [3H]ryanodine binding and
greatly increased phase 2 enhancement. DTT treatment reduced the
cross-links formed by diamide (Fig. 8A).
Therefore, the diamide-induced cross-links did not contribute to the
effects on [3H]ryanodine binding; instead, the oxidation
appeared to be protecting sulfhydryls during NEM alkylation.
As shown in Fig. 2, lowering the NEM concentration greatly slows the onset of the phase 2 and 3 effects, allowing us to look at a reaction that is primarily phase 1. To examine the effect of diamide pretreatment on the isolated phase 1 reaction, we pretreated membranes with 250 µM diamide and then examined the effect of 500 µM NEM on [3H]ryanodine binding (Fig. 7B). No phase 1 inhibition was seen with the diamide-pretreated membranes. These findings suggest that diamide is interacting with and protecting the phase 1 sulfhydryls. Alternatively diamide may be altering the conformation of the Ca2+ release channel, such that the phase 1 sulfhydryls no longer react as rapidly. To demonstrate this protection we pretreated membranes with 250 µM diamide, diluted the diamide to less than 25 µM, reacted with 5 mM NEM for 30 min on ice, and then reduced with 10 mM DTT, washed, and tested the effect of a second addition of 500 µM NEM on the binding of [3H]ryanodine. The reaction was again stopped with 10 mM DTT prior to the binding assay. These data are shown in Fig. 7C. As can be seen in this figure, reduction after diamide and NEM treatment restored the ability of NEM to inhibit [3H]ryanodine binding in a phase 1-like reaction. The initial binding and plateau binding to the membranes treated with diamide and then NEM was increased, as would be expected from membranes that have been alkylated at the phase 2 sites (see Fig. 7A).
To obtain additional evidence that diamide has not altered the ability of NEM to enhance binding in a phase 2 reaction, membranes were pretreated with diamide and with low concentrations of NEM under conditions that in the absence of diamide would have produced phase 1 inhibition. NEM was then added under conditions determined to produce phase 2 enhancement (Fig. 7D). Phase 2 enhancement is not blocked by diamide pretreatment.
To demonstrate that the phase 1 reaction with NEM protects the sulfhydryls involved in the diamide cross-link, we assessed the ability of NEM in the different phases to block dimer formation. The steps in this experiment were: 1) incubation of membranes with 5 mM NEM for different periods of time, 2) reduction with DTT, 3) removal of DTT, and 4) cross-linking with 250 µM diamide. NEM at the earliest incubation times blocked dimer formation (Fig. 8B). If dimer formation is truly protecting sulfhydryls from the phase 1 reaction, it should be possible to reduce diamide-formed disulfides after the phase 2 NEM reaction and then reform dimers with diamide. The experiment involved the following steps: 1) incubation of membranes with 250 µM diamide, 2) treatment with NEM for different periods of time, 3) reduction with DTT, 4) removal of DTT, and 5) cross-linking with diamide. Dimers could reform if, prior to NEM and subsequent DTT treatment, the sulfhydryls were protected by diamide cross-linking (Fig. 8C).
The phase 1 effects on [3H]ryanodine binding correlated
well with the rate of incorporation of [14C]NEM into the
565-kDa band of the RYR1. This is demonstrated in Fig.
9, where membranes with and without diamide pretreatment were incubated with 1 mM [14C]NEM for
increasing periods of time. The 565-kDa band from a Coomassie Brilliant
Blue-stained gel was excised, digested as described under
"Experimental Procedures," and the radioactivity in the bands was
quantitated by liquid scintillation counting. The data were fit as the
sum of two exponentials, with the rapid labeling component having a
kobs of 0.26 min1 and the slower
component having a kobs of 0.067 min
1. From the fits it was determined that diamide
pretreatment reduced the fast phase by 81% and the slow phase 13%. In
these experiments the incorporation of [14C]NEM into the
565-kDa band under phase 1 conditions (the fast component of labeling)
reached a maximum of about 13.5 pmol/sample. The quantity of
[3H]ryanodine binding sites applied to each well of this
gel was 0.5 pmol, and, since 4 subunits produce a single binding site, this would correspond to 2 pmol of the RYR1 subunits (assuming that all
of the RYR1 can bind ryanodine). However, in this membrane preparation
32% of the RYR1 was in the 400-kDa calpain-derived fragment and,
therefore, each well should have about 1.4 pmol of the 565-kDa band.
Since there are 100 cysteines/subunit, the [14C]NEM
labeled about 10% of the RYR1 sulfhydryls under phase 1 conditions or
less than 10 cysteines/subunit. This is in reasonable agreement with
direct labeling experiments, where the incorporation of radiolabeled
NEM under phase 1 conditions is 10% (n = 2) of that
which is incorporated into the 565-kDa band with 1 mM
[14C]NEM in SDS sample buffer. For comparison the effect
of unlabeled NEM under these same conditions on
[3H]ryanodine binding is also shown in this figure. The
rapid phase of [14C]NEM labeling appears to correlate
with the phase 1 inhibition of [3H]ryanodine binding, and
this labeling is partially blocked by diamide pretreatment.
Redox modulation of excitation-contraction coupling is important physiologically. In the native state, intact skeletal muscle fibers produce detectable levels of both reactive oxygen species (12) and NO derivatives (13, 14). Oxidant depletion alters contractile function. In unfatigued muscle, selective scavenging of reactive oxygen species depresses force generation (12); inhibition of NO synthesis has the opposite effect, increasing force (13). Strenuous contractile activity accelerates the rate at which myocytes produce free radicals, and other reactive oxidants, e.g. superoxide anion radicals (16), hydroxyl radicals (17), and NO derivatives (14). Oxidants accumulate in the active muscle and contribute directly to the loss of contractile function that occurs in muscular fatigue (15). Oxidative effects on both unfatigued and fatigued muscle are consistent with an increase in cytoplasmic Ca2+ concentrations due to activation of the sarcoplasmic reticulum Ca2+ release channel (21). An important role of sulfhydryl groups in the modulation of the activity of the skeletal muscle Ca2+ release channel has been demonstrated by several laboratories (3-9, 27, 28). Both oxidizing compounds (3-9) and reagents that modify free sulfhydryls (27, 28) alter the activity of Ca2+ release channel.
To explore these phenomena we choose NEM, a reagent that alkylates cysteine residues, and diamide, a sulfhydryl oxidizing agent that cross-links near neighbor cysteine residues. NEM shows a three-step effect on the activity of the Ca2+ release channel; first it inhibits, then it activates, and finally it again inhibits the channel reconstituted into planar lipid bilayers. In general, agents that activate the Ca2+ release channel increase the apparent affinity of the protein for [3H]ryanodine, while those that inhibit channel activity decrease the apparent affinity (36). Consistent with this, NEM produces a similar three-phase effect on [3H]ryanodine binding: phase 1 inhibition, phase 2 enhancement, and phase 3 inhibition. As shown by the effects on [3H]ryanodine binding and channel activity, and by direct [3H]NEM labeling, sulfhydryls on the Ca2+ release channel are reacting sequentially with NEM to alter the structure and function of the protein.
Diamide activates the channel reconstituted into planar lipid bilayers and enhances [3H]ryanodine binding by increasing apparent affinity. The observation that either an oxidizing or an alkylating reagent can activate the channel raised the question of whether the activation was due to the loss of a free sulfhydryl. If this were true, we would expect the activation by the two reagents to involve the same cysteine residues. This is not the situation for diamide and NEM; instead, to activate the channel, NEM and diamide appear to react with different sulfhydryls. This conclusion is based on a number of observations: 1) NEM can further stimulate the activity of the diamide-activated single channel, 2) diamide blocks the phase 1 inhibitory effects of NEM on [3H]ryanodine binding, 3) phase 1 alkylation by NEM blocks dimer formation by diamide, and 4) diamide pretreatment enhances the activating effects of NEM on [3H]ryanodine binding. When phase 1 sulfhydryls are protected by diamide cross-linking, NEM alkylation markedly enhances [3H]ryanodine binding. Our data are most consistent with a model in which the activation by diamide is due to cross-linking of phase 1 sulfhydryls while the NEM activation is associated with alkylation of phase 2 sulfhydryls. Without actually identifying the cysteine residues involved in phase 1 alkylation and those involved in diamide cross-linking, it is impossible to totally eliminate the possibility that diamide is having a long distance effect on phase 1 sulfhydryls. However, extensive reaction with NEM after diamide cross-linking fails to prevent the recovery of the phase 1 effects of NEM after reduction of the cross-linked sulfhydryls, a finding that strongly supports our model of phase 1 sulfhydryls being at subunit-subunit contact domains. The effects of oxidation, reduction, and alkylation on the binding of [3H]ryanodine and on channel activity are summarized in Table I. Intersubunit cross-links are likely to involve different cysteines on the two adjacent subunits. We have not yet determined whether both of these sulfhydryls are alkylated by NEM in the phase 1 reaction. The second sulfhydryl may be alkylated at a later stage, or it may not react with NEM. A completely different sulfhydryl could be alkylated in phase 2. The sequentially reacting cysteine residues could be at different locations in the primary sequence of each subunit or could be the same residues on different subunits, reacting at different rates as a result of conformational changes in the protein. These issues remain to be resolved.
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Diamide-induced cross-links are detected in intact membranes, in detergent-solubilized membranes, and in purified ryanodine-binding proteins,3 suggesting that the cross-links are occurring between subunits of a tetramer. Extended incubations with diamide lead to the formation of higher molecular weight oligomers, which could be trimers or tetramers. Formation of higher oligomers suggests either that the cross-link involves different sulfhydryls on adjacent subunits or that there is more than one type of cross-link formed. The partners in the formation of the cross-linked complexes were identified by two-dimensional electrophoresis. The membrane preparation used for these studies contained a significant amount of a 400-kDa band, which has been identified previously as a proteolytic fragment of the Ca2+ release channel (38, 39). Experiments have been performed with membrane preparations that have very little proteolysis and membranes that were proteolyzed by endogenous calpain. The effects of NEM and diamide on the binding of [3H]ryanodine was unaffected by this proteolytic event. Both the 565-kDa full-length ryanodine-binding protein subunit and the 400-kDa fragment appear to be cross-linked by diamide to form dimers, suggesting that the site of the intersubunit cross-link is within the 400-kDa fragment. Very little cross-linking, however, occurs between the 400-kDa and the 565-kDa bands. This surprising finding suggests that the proteolytic events that produce the 400-kDa fragment are nonrandom. The most frequent neighbor of a 400-kDa proteolyzed subunit within a tetramer is another 400-kDa subunit, while the most frequent neighbor of the full-length 565-kDa protein is another 565-kDa protein. The proteolytic event is presumably due to the action of endogenous calpains (38, 39). One interpretation of these findings is that some of the Ca2+ release channel tetramers are by some means targeted for proteolysis such that all of the subunits in the protein are proteolyzed simultaneously. Other tetramers contain all intact subunits. This would suggest that some modification of the Ca2+ release channel allows it to be recognized by endogenous calpains. A second possibility is that once calpain binds to the tetramer it remains bound until all of the subunits are cleaved. We are currently investigating these intriguing possibilities.
Cross-linking of sulfhydryls by diamide activates the channel, indicating that sulfhydryls within this class may be the targets of redox modulation of the Ca2+ release channel and that these redox-sensitive sulfhydryls are located in domains where subunits contact one another. There is no indication of the involvement of any other protein in the disulfide bond formation induced by diamide. This is in contrast to the results of Liu et al. (22, 23), who suggest that triadin is cross-linked to the Ca2+ release channel by oxidizing agents. We were unable to find evidence of such a cross-link.
In summary, we demonstrate the existence of at least three classes of functionally important sulfhydryls on the Ca2+ release channel, modification of which alter channel activity and [3H]ryanodine binding (Table I). Both NEM and diamide can activate the Ca2+ release channel, but the reactions involve different sulfhydryls. Diamide cross-links subunits within the tetramer. This cross-linking appears to correlate with channel activation and with oxidation of a class of sulfhydryls, which, in the absence of diamide, react rapidly with NEM to inhibit the channel. Alkylation of these phase 1 sulfhydryls prevents dimer formation. The phase 1 sulfhydryls, therefore, may be located at contact domains between subunits. These studies will be useful in designing strategies to differentially label phase 1 and phase 2 sulfhydryls to map their location in the primary sequence of the Ca2+ release channel. This may also enable us to define some of the parts of the protein that are in regions of subunit-subunit contact in the three-dimensional structure of the protein.
We thank Lynda Attaway, Shu-Jun Dou, Dr. Barbara Williams, Dr. SiQi Liu, and Dr. Dolores Needleman for advice and assistance in preparing this manuscript.