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Biophys J, December 1999, p. 3010-3022, Vol. 77, No. 6
*Muscle Research Group, John Curtin School of Medical Research, Canberra, ACT 2601, Australia, and #Second Institute of Physiology, University of Heidelberg, Heidelberg D69120, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyRMH (Arg615 to Cys615) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4,4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyRMH channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys615 in RyRMH is functionally unimportant in redox cycling.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Contraction of striated muscle depends on
Ca2+ release from the sarcoplasmic reticulum (SR), through
ryanodine receptor (RyR) calcium release channels. Oxidants are
important ligands in the physiological activity of RyRs and act by
oxidation of free sulfhydryl (SH) groups, because their effects are
prevented by sulfhydryl reducing agents (Boraso and Williams, 1994
;
Favero et al., 1995; Abramson et al., 1995). Oxidants activate and can
inhibit RyRs (Holmberg et al., 1991; Holmberg and Williams, 1992;
Boraso and Williams, 1994; Favero et al., 1995; Stoyanovsky et al.,
1997). Oxidation-induced activation proceeds under in vivo conditions, in the presence of glutathione (GSH) (Koshita et al., 1993), and is
enhanced during ischemia or reperfusion, in which oxygen free radicals
increase and the ratio of GSH:GSSG falls (Sies et al., 1972;
Curello et al., 1985).
Reagents that react specifically with free SH groups are used as model
compounds to look at the effects of oxidation and the importance of
cysteine residues in RyR channel function. Reactive disulfides 4,4'- or
2,2'-dithiodipyridine (4,4'-DTDP or 2,2'-DTDP) activate and then block
cardiac RyRs when applied to the cytoplasmic (cis) side of
the channels (Eager et al., 1997; Eager and Dulhunty, 1998). Skeletal
RyRs have also been reported to be activated, but not inhibited, by
cis 4,4'- or 2,2'-DTDP and by GSSH (Nagura et al.,
1988; Marengo et al., 1998) and to be inhibited by the reducing agents
GSH, dithiothreitol (DTT), or 
-mercaptoethanol (BME). The domain of
the RyR on which oxidants act is not clear. Both lipid-soluble and
water-soluble reagents can partition into the membrane, to a greater or
lesser extent, depending on their pKa values and could
react with -SH groups on either the side to which they are added or in
the transmembrane parts of the RyR protein. It is unlikely that
reagents would act rapidly on the opposite side of the bilayer, because
reagent crossing the bilayer would be diluted in the large volume of
the opposite solution and would take a long time to reach activating
concentrations. Water-soluble oxidants appear to react with SH groups
only in the cytoplasmic or transmembrane domains of the mammalian RyRs. Methanethiosulfonate derivatives block skeletal muscle RyRs only from
the cis side (Quinn and Ehrlich, 1997). Some residues
(-aSH and -iSH) responsible for activation or
inhibition of cardiac RyRs are accessible to thimerosal from either
side of the bilayer and are located in the transmembrane domain, while
other activating (-a*SH) residues are confined to the
cytoplasmic domain (Eager and Dulhunty, 1999). There have been no
similar reports of the long-term effects of thiol-specific oxidizing
agents added to the luminal or cytoplasmic sides of mammalian skeletal
RyR channels.
The present study examines the effects on single skeletal RyRs of long
exposure to 2-10 mM concentrations of the thiol-specific 4,4'-DTDP or
5,5'-dithiobis-(2Nitrobenzoic acid) (DTNB) or DTT. The experiments
tested the hypothesis that, as in cardiac RyRs (Eager et al., 1999),
three classes of -SH (-aSH and -iSH, within the
transmembrane domain, or -a*SH in the cytoplasmic domain) are available in skeletal RyRs for oxidation by specific sulfhydryl reagents. It was possible that the same thiols could be oxidized in
cardiac and skeletal RyRs, because 71 of >80 cysteine residues are
conserved between the two isoforms (Otsu et al., 1990). The experiments
tested an additional hypothesis that the
Arg615-to-Cys615 substitution in
RyRMH from pigs susceptible to malignant hyperthermia (MH)
provides an additional -SH group for oxidation. MH is an inherited
skeletal muscle disorder, characterized by increased Ca2+
release from SR (Ohta et al., 1989; Carrier et al., 1991; Mickelson and
Louis, 1996) and reduced inhibition of RyRs by Ca2+ (Fill
et al., 1991; Shomer et al., 1993) or Mg2+ (Laver et al.,
1997).
Novel findings are 1) that the water-soluble DTNB (in either
cis or trans solutions) and the lipid-soluble
4,4'-DTDP (in the cis solution) activate skeletal RyRs in a
similar way; 2) 4,4'-DTDP added to the cis solution, but not
DTNB (cis or trans), inhibits skeletal RyRs after
>5 min; 3) in the absence of an oxidizing reagent, 10 mM DTT activates
skeletal RyRs from the cis or trans solution; 4)
"control-like" channel activity is maintained when 1 mM DTNB and 10 mM DTT are present together on either the same or opposite sides of the
bilayer; and 5) effects of oxidation and reduction are the same in
normal RyRs and RyRMH. Our conclusions are that 1)
-aSH is located in the skeletal RyR transmembrane domain,
as in cardiac RyRs; 2) -a*SH, in cardiac RyRs, is not available for oxidation in skeletal RyRs; 3) a modified thiol in the
transmembrane domain (-abS-R, where R is either a protein S
if the modified thiol is a disulfide, or an N if the modified group is
nitrosylated; Xu et al., 1998) normally suppresses skeletal RyR
activity; and 4) the Arg615-to-Cys615
substitution in MH does not provide an additional SH group for oxidation by thiol-specific reagents.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Biological material and caffeine-halothane contracture test for MH susceptibility
The methods for genetic testing, muscle dissection,
caffeine-halothane contracture testing, preparation of SR vesicles, and single-channel recording have been described previously (Otsu et al.,
1992; Owen et al., 1997; Laver et al., 1997). Muscle and blood samples
were obtained from three homozygous normal pigs (one Belgium Landrace
and two Landrace) and three homozygous MH pigs (two Belgium Landrace
and one Landrace) aged ~4 months. Each animal was genetically tested
for a normal or MH RyR allele (containing either Arg615 or
Cis615). The SR preparations were from the same animals as
those used in Laver et al. (1997). The description of anesthetic
techniques, muscle dissection, and halothane/caffeine contracture tests
are given by Laver et al. (1997). All fiber bundles from the three homozygous normal animals failed to respond to halothane or 2 mM
caffeine, while all fiber bundles from the three homozygous MH animals
developed tension in response to both drugs.
Isolation of SR vesicles
The preparation of crude SR vesicles was based on the methods of
Meissner (1984) and Ma et al. (1995). The freshly dissected back and
leg muscle was washed in cold phosphate-buffered saline containing 2 mM
EGTA (pH 7.0), trimmed of fat and connective tissue, cubed, and either
frozen in liquid N2 and stored at 
70°C or freshly processed. The fresh or thawed muscle cubes were suspended in (mM) 5 Tris maleate, 100 NaCl, 2 EDTA, 0.1 EGTA (pH 6.8) (5 ml/g of tissue).
The muscle was homogenized in a Waring blender in four 15-s high-speed
bursts. The homogenate was centrifuged at 2600 × g for
30 min, and the supernatant ws filtered through cotton gauze and
centrifuged at 10,000 × g for 30 min. The pellet (P2) was collected, and the supernatant was centrifuged again at 35,000 × g and the pellet (P3) collected. Pellets P2 and P3 were
resuspended in (mM) 5 Tris-2-(N-morpholino)ethanesulfonic
acid (Tris-MES), 300 sucrose, 100 KCl, 2 DTT (pH 6.8). Aliquots of the
suspensions were frozen in liquid nitrogen and stored at 70°C. All
buffers contained the protease inhibitors phenylmethylsulfonyl fluoride (0.7 mM), leupeptin (1 µg/ml), pepstatin A (1 µM), and benzamidine (1 mM).
Lipid bilayer techniques
The lipid bilayer and single-channel recording technique are
described by Ahern et al. (1994) and Laver et al. (1995). Bilayers were
formed from phosphatidylethanolamine, phosphatidylserine, and
phosphatidylcholine (5:3:2 w/w) (Avanti Polar Lipids, Alabaster, AL)
across an aperture with a diameter of 200-250 µm in the wall of a
1.0-ml Delrin cup (Cadillac Plastics, Australia). TC vesicles (final concentration 10 µg/ml) were added to the cis
chamber and stirred until vesicle incorporation was observed. The
cytoplasmic side of channels incorporated into the bilayer faced the
cis solution. The bilayer potential was controlled and
single-channel activity was recorded with an Axopatch 200A amplifier
(Axon Instruments, Foster City, CA). For experimental purposes, the
cis chamber was held at ground and the voltage of the
trans chamber was controlled. Bilayer potential is expressed
in the conventional way as Vcis Vtrans (i.e.,
Vcytoplasm Vlumen).
Bilayers were formed and vesicles incorporated into the bilayer, using cis and trans solutions containing (mM) 230 Cs methanesulfonate (CsMS), 20 CsCl, 0.1 CaCl2, and 10 N-tris[hyroxymethyl]methyl-2-aminoethanesulfonic acid (TES) (pH 7.4 adjusted with CsOH). The cis solution also contained 500 mM mannitol to aid SR vesicle fusion and RyR incorporation into the bilayer.
Recording and analysis of single-channel data
Channel activity was recorded at 40 mV. In general activity
was recorded for a 2-min control period before and 1 min after 0.5-s
voltage pulses to +40 mV. Drugs were then added to either the
cis or trans chamber with a ~10-s stirring
period, and then activity was recorded for several minutes. Voltage
pulses to +40 mV were occasionally applied to the bilayer after drug
addition to determine whether channels could be activated by the change in bilayer potential. Because RyR channel activity can increase immediately after a voltage pulse (Zahradnikova and Meszaros, 1998;
Laver and Lamb, 1998), the first 30 s after each voltage pulse was
excluded from analysis.
Channel activity was filtered at 1 kHz (10-pole low-pass Bessel, 3
dB) and digitized at 2 kHz. Analysis of single-channel records (using
Channel 2, written by P. W. Gage and M. Smith) yielded channel
open probability (Po), frequency of events
(Fo), open times, closed times, and mean open or
closed times (To or Tc),
as well as mean current (I'). The open discriminator was set
at ~25% of the maximum current, and the closed discriminator at 50%
of the open discriminator, so that openings to both subconductance and
maximum conductance levels were included in the analysis. Single-channel parameters were measured during the 30 s, showing maximum I' during the control period and then the period of
maximum I' after the addition of drugs. Dwell-time
distributions were plotted as the frequency of openings in logged bins
(Sigworth and Sine, 1987) to display the large range of open times seen in control recordings and after the addition of oxidizing reagents. The
fit of a multiple exponential function to the data was assessed using a
least-squares fit.
Statistics
Average data are given as mean ± SEM. The significance of
the difference between control and test values was evaluated with a
Student's t-test, either one or two sided and for
independent or paired data, as appropriate. Differences were considered
to be significant when p 
0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of DTNB or 4,4'-DTDP on normal RyRs
Activation by DTNB
RyRs from homozygous normal pig muscle were activated by addition of the hydrophilic DTNB (1 mM) to either the cis or trans chamber. Control RyR activity was characterized by brief openings to the maximum conductance and to lower conductance levels (Figs. 1, A and B, and 2, A and B). Longer openings appeared with DTNB (Figs. 1 A and 2 B). Channel activity increased with a delay of ~1 min after DTNB was added to the cis chamber in 13 of 14 channels, with a delay of ~3 min after trans DTNB was added to 10 of 11 RyRs. Average mean current (I') increased from a control value of1.43 ± 0.12 pA to 3.91 ± 0.19 pA (n = 10) or from 2.73 ± 0.38 pA to
5.81 ± 0.64 pA (n = 6). When the average ratio
of mean current before and after addition of the drugs for individual channels was determined, there was an approximately fivefold increase with cis DTNB and an approximately threefold increase with
trans DTNB (Table 1). Note
that the delay was assessed by eye from channel records. The numbers of
channels for I' in Table 1 are fewer than the number for the
delay because I' was measured using Channel2 analysis of
low-noise recordings only.
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Activation by 4,4'-DTDP
4,4'-DTDP (1 mM cis added in ethanol, 1% v/v) caused an approximately sevenfold increase in I', which began ~2 min after its addition to 15 of 15 channels (Table 1). In contrast to sheep cardiac (Eager et al., 1997) or rabbit skeletal (Ahern et al., 1997a) RyRs, 1% ethanol (alone) increased I' after ~3 min
in eight of eight RyRs (Table 1). Therefore 4,4'-DTDP was dissolved in cis solution at 10 mM and added to the cis
chamber to a final concentration of ~1 mM. In this situation,
I' increased in 18 of 21 channels, after a delay of ~1.5
min (Table 1). Activity returned toward control after 2 mM
cis DTT addition to each of four single 4,4'-DTDP-activated
RyRs (Figs. 1 B and 2 A). Thus 4,4'-DTDP also
activated RyRs via oxidation of -SH groups, which could also be
-aSH in the transmembrane domain.
Inhibition by 4,4'-DTDP
Cis 4,4'-DTDP (1 mM) abolished activity in seven of 10 RyRs, after 334 ± 52 s (4,4'-DTDP plus ethanol, Fig. 2 AIII), or in five of nine channels after 456 ± 82 s (4,4'-DTDP added in cis solution). Inactivated channels were not reactivated by four pulses to +40 mV (0.03 Hz). Infrequent activity remained in seven of 19 channels, for 26 min in one case. RyRs were not inactivated after 5-30 min with 1% ethanol alone (n = 9). Cis DTT (2 mM) restored activity after 129 ± 62 s in three of five RyRs inactivated by 4,4'-DTDP plus ethanol (Fig. 2 AIV) or after 240 ± 165 s in three of five RyRs inactivated by 4,4'-DTDP alone. Recovery with DTT shows that inactivation is due to -SH oxidation and that -iSH is present in skeletal RyRs and accessible to DTT. In contrast to 4,4'-DTDP, 1 mM DTNB (either cis or trans) did not inactivate RyRs after 5-20 min (Fig. 2 B, II and III). If activity fell, it increased again after pulses to +40 mV.Comparison of RyR and RyRMH
The characteristics of normal RyRs and RyRMH (from
homozygous MH pigs) were similar under control conditions at 40 mV,
with 100 µM Ca2+ and symmetrical 250 mM CsMS (Fig.
3). Single-channel conductance was
452 ± 9 pS (n = 4) for normal RyRs and 470 ± 11 pS (n = 5) for RyRMH, and control
I' for RyRMH was 1.76 ± 0.13 pA
(n = 39), compared with ~2.1 pA for 16 normal RyRs
(Results above). In addition, both types of channel were 1) locked into
a submaximum conductance state with 10-15 µM cis
ryanodine (RyR, n = 11; RyRMH,
n = 12), 2) blocked by 5 µM cis ruthenium
red (RyR, n = 3; RyRMH, n = 2), and 3) activated by 5 mM ATP (RyR, n = 4;
RyRMH, n = 5).
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Effects of reactive disulfides on RyRs from MH pigs
Activation by DTNB or 4,4'-DTDP
Activity increased fourfold in 14 of 14 RyRMH channels ~3 min after the addition of 1 mM cis DTNB or approximately fourfold in 16 of 17 channels ~2 min after trans DTNB (Fig. 3, Table 1). Activation of RyRMH by cis DTNB was significantly slower than activation of normal RyRs (p < 0.01). There were no other differences in activation by DTNB between normal RyRs and RyRMH. 4,4'-DTDP added to RyRMH, with ethanol or alone (in cis solution), induced three- to fourfold increases in average I' (Fig. 3), after 2.5-3.5 min, while ethanol alone caused an approximately twofold increase in I' after ~2.5 min (Table 1). RyRMH activity returned to control levels within 1 min after 10 mM DTT was added to the cis side of channels activated by cis DTNB (four of four) or trans DTNB (three of three) or after 2 mM cis DTT was added to cis 4,4'-DTDP-activated channels (five of five). The similar results with normal RyRs and RyRMH show that the Arg615-to-Cys615 substitution in MH does not alter the ability of sulfhydryl-specific reagents to activate RyRs by oxidizing -aSH. The differences between RyRMH and normal RyRs in the rate of activation by cis DTNB could be explained by structural changes that reduce the accessibility of -aSH in RyRMH.Inhibition of RyRMH
RyRMH channels were inactivated after 458 ± 30 s exposure to 1 mM cis 4,4'-DTDP (added with 1% ethanol, n = 10), and nine of the 10 channels were reactivated 160 ± 30 s after adding 2 mM DTT. Similarly, 1 mM cis 4,4'-DTDP added alone (in cis solution) abolished RyRMH activity in three of six channels after 383 ± 156 s, and the three channels recovered 50 ± 26 s after 2 mM DTT was added. No RyRMH channels were inhibited during 3.5-32-min exposure to DTNB (cis or trans, n = 13) or 4-30-min exposure to 1% cis ethanol (n = 5). Thus inhibition of RyR channels via oxidation of -iSH was not altered by the MH mutation. There were some curious exceptions to the general observations reported above. One normal and two RyRMH channels were inactivated 2-7 min after 4,4'-DTDP was added, without initial activation, and inhibition was relieved 40-90 s after cis DTT was added. This confirmed independent channel activation and inhibition and supported separate -aSH and -iSH residues (see also Eager et al., 1998). Activity in three RyRMH and two
normal RyRs fell after initial activation by cis 4,4'-DTDP,
and activity in two normal RyRs fell 5 min after cis
4,4'-DTDP was added, without any initial activation. There was no
further change in activity in these seven channels when 2 mM
cis DTT was added, suggesting that the disulfide formed upon
exposure to 4,4'-DTDP (-aS-Sr, where -Sr is contributed
either by the reactive disulfide or by the channel protein) was reduced during exposure to 4,4'-DTDP and hence that the affinity of
-aSH for 4,4'-DTDP in these channels was less than normal.
Single-channel properties of RyR and RyRMH
There was no significant difference between normal RyR (n = 48) and RyRMH (n = 45) in the steady-state parameter values for Po, Fo, To, and Tc measured over 30-s periods (Table 2). The two types of channel showed similar modes of activity, transitions between modes and responses to voltage pulses to +40 mV (Fig. 4). Predominant modes were either low activity or high activity, unaltered after the voltage pulse (Fig. 4, I and II) or voltage-activated increase in activity immediately after the voltage pulse, which then decayed after 10-20 s to a lower level that was also seen before the voltage pulse (Fig. 4 III). Strong submaximum conductance activity was also seen in both normal RyRs and RyRMH (Fig. 4, C and D), giving an average open channel conductance that was 0.50 ± 0.05 of the maximum conductance in normal RyRs or 0.37 ± 0.09 in RyRMH.
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Effects of oxidizing reagents and ethanol on single-channel parameters of normal RyRs and RyRMH
The oxidizing reagents increased mean open time (To) in all normal RyRs and all RyRMH channels, with significant increases in average To under most conditions (Table 2). The effect on other aspects of channel activity was complex, with an increase in mean closed time (Tc) in some channels and a decrease in Tc in others, so that the changes in average Fo and Po were not significant (Table 2). DTT at 10 mM reversed the effects of DTNB, and at 2 mM reversed the effects of 4,4'-DTDP in normal RyRs and RyRMH. Data were pooled from the few channels that were suitable for analysis and were exposed first to an oxidizing reagent and then to DTT. I', Po, and To fell significantly after DTT was added to the oxidation-activated channels (Fig. 5). The similar actions of cis DTNB, trans DTNB, and cis 4,4'-DTDP on the single-channel parameters during activation and the similar reversal of these actions by DTT provide further evidence that one class of cysteine residues (-aSH) is oxidized in each of the three situations.
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Effects of DTNB and 4,4'-DTDP on open and closed time distributions
Open times for normal RyR and RyRMH fell into two
exponential components under control conditions and three exponential
components after oxidation-induced activation (Figs.
6 and 7).
Closed times for both normal RyRs and RyRMH channels fell
into three exponential components under control conditions and after
oxidiation-induced activation. Data for normal RyRs and
RyRMH were combined (Fig. 7) because there were no
consistent differences in the average time constants or probability of
events between the two channel types. Under control conditions and
during oxidation-induced activation, the shortest open time constant,
o1, was ~1-2 ms, and the second time constant,
o2, was 7-13 ms. The longest time constant,
o3, was seen only in "activated" channels; it was
50-100 ms and contained <10% of openings. The increase in
To was largely due to the appearance of
o3.
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Closed time constants under control and oxidation-induced activation
conditions were c1 (2-4 ms), c2 (9-23
ms), and c3 (90-480 ms) (Figs. 6 and 7). The longest
component (c3) contained very few events and did not
change in any consistent way during oxidation-induced activation. The
similar open and closed time distributions with cis DTNB,
trans DTNB, and cis 4,4'-DTDP are consistent with
the hypothesis that the same cysteine residues are oxidized with each
reagent. An additional fourth long open time constant in cardiac
RyRs exposed to cis 4,4'-DTDP (Eager et al., 1999) was not
seen in skeletal RyRs.
An effect of oxidizing reagents on burst behavior
Channel openings in 20-50% of normal or RyRMH channels under control conditions was continuous, while activity in the other channels was clustered into bursts, separated by closures of 0.5-60 s (not included in Figs. 6 and 7). Continuous or burst activity was observed in each of the three modes (high, low, or voltage-activated activity) defined in the text description of Fig. 4 (I-III) above. When either 1 mM DTNB or 1 mM 4,4'-DTDP was added to the cis chamber, 97% of channels adopted burst behavior, in addition to the increase in channel open time (Table 3). Only 59% of normal RyRs or RyRMH had burst behavior with trans DTNB-induced activation, or 45% during activation with cis or trans DTT (10 mM, Results below) or with ethanol (Table 3). The stabilization of burst behavior by oxidizing reagents in the cis chamber was not reversed by DTT. Bursting behavior remained in 94% of channels when 10 mM DTT was added to the cis or trans bath after 1 mM cis DTNB, and remained in 50% of channels when 10 mM DTT was added to the trans bath after 1 mM trans DTNB.
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This effect of oxidizing reagents on burst activity suggested that DTNB and 4,4'-DTDP modified a site on the cytoplasmic side of the channel that alters channel gating to stabilize bursting behavior. It is unclear whether the effect is due to -SH oxidation or an interaction between the oxidizing reagents and other sites on the channel, because the effect was not reversed by DTT.
Further evidence that -aSH is located in the transmembrane domain
If the hypothesis that -aSH is located in the transmembrane domain and is accessible from either side of the bilayer is correct, then activation by DTNB on one side of the bilayer should be reversed by adding DTT to the opposite chamber. In this experiment, five of six RyR channels were activated when DTNB was added to the cis chamber, and activity fell in five of the channels when DTT was added to the trans chamber. Conversely, one of three other channels was activated when DTNB was added to the trans chamber, and activity fell in that channel when DTT was added to the cis chamber.
Evidence for a modified thiol in the transmembrane domain
DTT at 2 mM, added in the absence of an oxidizing reagent, did not significantly alter RyR activity. The mean current, normalized to control, was 3.6 ± 2.8 in five normal RyRs after 2 mM DTT addition and 2.7 ± 0.4 in five RyRMH channels. On the other hand, 10 mM DTT significantly activated normal RyRs and RyRMH when added to either the cis or trans chamber in the absence of added oxidizing reagent; activity then fell toward control levels when 1 mM DTNB was added to the opposite side of the bilayer (Fig. 8). Cis DTT (10 mM) activated five of five RyRs (two normal; three RyRMH), while trans DTT activated two of four normal RyRs and three of three RyRMH. Data from normal RyRs and RyRMH is combined in Fig. 8. The two RyRs not activated by trans DTT were not included, because their control activity was high (I' of 4.0 and 6.8 pA) and outside the range of 0.2-1.2 pA in the other five channels. Average I' increased significantly with 10 mM DTT (cis or trans) and then fell significantly after DTNB addition to the opposite chamber (Fig. 9). Interestingly, activity in the two channels that were not activated by cis DTT fell to lower levels when DTNB was added to the trans chamber, with similar approximately sixfold reductions in I'. These channels may have already been in a reduced state before the addition of DTT.
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These results suggest that a class of modified thiol
(-abS-R-, where R is either a protein S if the modified
thiol is a disulfide, or a N if the modified group is nitrosylated; Xu
et al., 1998) is available to 10 mM DTT from either the cis
or trans chamber and that the reduced -abSH can
be oxidized by DTNB from the opposite side of the bilayer. The
accessibility from either side of the bilayer indicates that
-abS-R- and -abSH are located in the
transmembrane domain.
"Control-like" channel activity is retained in the presence of 1 mM DTNB plus 10 mM DTT
Channel activity returned to "control-like" levels when 10 mM
DTT and 1 mM DTNB were present on either the same or opposite sides of
the bilayer. This was confirmed in further experiments, in which the
"control-like" activity was maintained if 10 mM DTT was present on
both sides of the channel, with 1 mM DTNB on one side only (five of six
experiments with cis DTNB, or three of three with
trans DTNB). Average I' measured in four of the
channels with cis DTNB or two of the channels with
trans DTNB was 2.19 ± 0.32 pA. "Control-like"
activity was retained when 10 mM DTT was removed from one chamber,
leaving 10 mM DTT and 1 mM DTNB in the cis chamber in two
cases, or in the trans chamber in a third case
(I' = 1.33 ± 0.51 pA).
The assertion that channel activity in the presence of 10 mM DTT and 1 mM DTNB was "control-like" was supported by a final experiment
(n = 2) in which the trans chamber initially
contained 1 mM DTNB plus 10 mM DTT and the cis chamber
contained 1 mM DTNB (I' = 0.97 ± 0.39 pA). The
trans chamber was perfused with normal trans
solution, leaving 1 mM DTNB in the cis solution. In both cases, RyR activity increased after perfusion, as it usually did when
DTNB was present alone in the cis chamber (I' = 5.50 ± 0.33 pA).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We found that DTNB activated skeletal muscle RyRs from either the
cytoplasmic or luminal side of the channel, while 4,4'-DTDP activated
and then inhibited RyRs from the cytoplasmic solution. Activation by
both reagents and inhibition by 4,4'-DTDP were reversed by DTT and thus
are due to sulfhydryl oxidation. The results can be explained by
oxidation of two classes of sulfhydryl, -aSH in the
transmembrane domain for activation or -iSH in a
hydrophobic environment for inhibition. Separate -aSH and
-iSH residues have also been postulated for cardiac RyRs
(Eager and Dulhunty, 1998, 1999). Additional novel findings were that
1) addition of the oxidizing reagents to the cis side of
RyRs stabilized bursting channel activity, suggesting that cytoplasmic
residues regulate burst activity; 2) addition of 10 mM DTT to either
side of the channel caused activation, which was reversed when DTNB was
added to the opposite side, suggesting that a modified thiol
-abS-R- in the transmembrane domain normally inhibits
activity; and 3) "control-like" channel activity was maintained in
the presence of 1 mM DTNB and 10 mM DTT. Finally, the effects of the
oxidizing reagents on RyRs from normal and MH pigs were similar.
Activation of skeletal RyRs by reactive disulfides
Activation of skeletal RyRs by DTNB and 4,4'-DTDP confirms
previous reports on skeletal RyRs (Nagura et al., 1988; Marengo et al.,
1998; Zable et al., 1997). Similar activation, with a long time
constant component introduced into the open time distribution, is seen
in cardiac RyRs exposed to 4,4'-DTDP or thimerosal. An increase in open
frequency is also seen in cardiac RyRs. A fourth long time constant
component in the open time distribution of cardiac RyRs oxidized by
4,4'-DTDP was not seen in skeletal RyRs, suggesting that the
-a*SH class of sulfhydryl, postulated for the cardiac RyR
(Eager and Dulhunty, 1998), either is not present or is not available
for oxidation in skeletal RyRs. The fact that reversal of activation by
1 mM DTNB required 10 mM DTT, while reversal of activation by 4,4'-DTDP
or thimerosal requires 2 mM DTT (results above and Eager et al., 1999),
suggests that DTNB has stronger oxidative properties than 4,4'-DTDP.
The failure of GSSG to activate skeletal RyRs from the luminal solution
(Zable et al., 1997) might have been due to the weak oxidizing ability of GSSG.
What do similar changes to reagents added to either side of the RyR mean in terms of the location of target residues?
The ability of DTNB to activate RyRs from the luminal or
cytoplasmic side and the reversal of activation by 10 mM DTT added to the opposite side of the channel suggest that -aSH is
accessible to DTNB and DTT from the cytoplasmic and luminal solutions.
This accessibility to water-soluble reagents could suggest that target residues are located in the channel pore. However, DTT with a pKa of 9.0-10 (Shaked et al., 1980) would be largely
uncharged at pH 7.4 and would rapidly partition into the membrane.
Similarly, 1-10% of DTNB with a pKa of 5-6 (Houk
et al., 1987) would enter the membrane. Thus the water-soluble agents
could access residues located in the transmembrane domain, not in the
pore, although activating residues in a transmembrane, rather than pore
location, would see only 10-100 µM DTNB. This is not an unreasonable
[DTNB] for activation, because DTNB is a strong oxidizing reagent
(above), and NO at ~40 nM nitrosylates thiol groups and activates
skeletal RyRs (Hart and Dulhunty, unpublished observations), while
cardiac RyRs are activated by 100 µM 4,4'-DTDP (Eager et al., 1997).
It is unlikely that the reagents, either crossing the membrane or passing through the pore, could have targeted residues located on the opposite side of the channel and remote from the membrane, because dilution in the large volume of solution would mean that it would take a long time for their concentrations to increase to active levels. Thus activation with DTNB or recovery with DTT would have been faster when the reagents were applied to the side of the channel containing the residues. Because similar rates of activation and deactivation were seen with cis and trans applications of both reagents, we conclude that DTNB and DTT act at a transmembrane (possibly pore) location.
Another possibility is that separate actions of DTNB on cytoplasmic and
luminal sulfhydryls result in similar functional effects on RyR
activity (Eager et al., 1998). However, reversal of activation by DTT
on the opposite side of the bilayer would not be expected if
-aSH were distributed over the luminal and cytoplasmic
domains of the RyR. If -aSH is located in the channel pore,
then DTT, DTNB, and 4,4'-DTDP must penetrate the channel. Molecules of
a mass similar to that of DTT (formula weight, FW 154) and 4,4'-DTDP (FW 220), such as glucose and xylose (FW 180 and 150, respectively), pass slowly through skeletal RyRs (Meissner, 1986; Kasai et al., 1992).
DTNB (FW 396) and thimerosal (FW 405) have a greater mass but may
nevertheless assume conformations that allow them to also pass through
the channel. Curiously, neither DTNB nor 4,4'-DTDP nor DTT blocks the
channel into the low-conductance states seen in the presence of the
smaller (FW 110-172) methanethiosulfonate (MTS) derivatives (Quinn and
Ehrlich, 1997). Although DTNB and 4,4'-DTDP are cleaved during
oxidation of protein thiols, their half-masses of 110 and 198 remain
equivalent to that of the MTS compounds. If DTT, DTNB, and 4,4'-DTDP
enter the pore and interact with sulfhydryl residues to alter channel
gating, they must do so without physically blocking the pore.
Inhibition of skeletal RyRs by reactive disulfides
We propose that oxidation of separate -aSH and -iSH leads, respectively, to activation and inhibition of RyRs, rather than oxidation of one class of sulfhydryl that first activates and then inhibits the channel. Evidence for separate residues is that 1) activation by DTNB was not followed by inhibition and 2) inhibition was observed without preceding activation in some channels (see also Eager et al., 1997). RyR inhibition by 4,4'-DTDP, but not DTNB, suggested that -iSH may be located in a domain of the
skeletal RyR that is inaccessible to DTNB. In contrast, the
water-soluble thimerosal inhibited cardiac RyRs (Eager et al., 1999).
This apparent difference may have been due to the use of thimerosal in
cardiac and DTNB in skeletal RyRs, because thimerosal penetrates
proteins more effectively than other thiol reagents (van Iwaarden et
al., 1992).
An alternative possibility is that the different abilities of DTNB,
4,4'-DTDP, and thimerosal to inhibit RyRs are related to their
different redox potentials. Sulfhydryl-specific oxidizing reagents can
react with proteins in two steps (Glazer, 1970). The first step is the
formation of a mixed disulfide between the protein thiol
(-aSH, for example) and the reagent (rS-Sr):
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Comparison between cardiac and skeletal RyRs
It is possible that the same cysteine residues form
-aSH in cardiac and skeletal RyRs. Many cysteine residues
in the channel domain are conserved between cardiac and skeletal RyRs
(Otsu et al., 1990) and are in an appropriate location for
-aSH. The subtly different effects of oxidation on the
gating of the cardiac and skeletal RyRs could be attributed to a
difference in the connections between the -aSH and the
channel gating mechanisms. Such a difference may be imposed by sequence
differences between the cardiac and skeletal RyRs (having only 66%
sequence identity; Otsu et al., 1990), which could impose structural
differences between the proteins.
Skeletal RyR channels recovered from inhibition when DTT was added to
the cis solution, but the loss of activity in cardiac RyRs
could not be reversed by DTT (Eager et al., 1997). Inhibition of
skeletal RyRs by NO is also relieved by DTT (Hart and Dulhunty, unpublished observations). These observations suggest that the oxidized
xS-Si- (where -xS is contributed either by DTNB or
4,4'-DTDP or by the protein) is more accessible to DTT in skeletal RyRs than in cardiac RyRs.
Effect of DTT alone on pig skeletal RyRs
Addition of 10 mM DTT to either side of the bilayer (with oxidizing reagents absent from cis and trans solutions) increased RyR activity. Increased mammalian skeletal RyR activity with DTT is in contrast to a fall in Po when 20 mM GSH was added to frog skeletal or rabbit cardiac RyRs (Marengo et al., 1998) or when 10 mM DTT was added to sheep cardiac
RyRs (Eager et al., 1997). Zable et al. (1997) also showed a reduction
in rabbit skeletal RyR activity with 6-10 mM GSH and reduced ryanodine
binding with GSH, DTT, and BME. The reason for the difference between
our results and those of Zable et al. may be partly due to sequence
differences between pig and rabbit RyRs (see below). However, we also
find that rabbit skeletal RyR activity increases when 2 mM DTT alone is
added to the trans chamber (Green, Hart, and Dulhunty,
unpublished observations). In agreement with our findings, DTT induces
contraction in intact frog skeletal muscle fibres by triggering
Ca2+ release from SR (Oba et al., 1996).
Activation of skeletal RyRs by cis or trans DTT
and its reversal with DTNB in the opposite chamber suggest that a class
of modified thiols, -abS-R-, which normally suppresses
channel activity, is present in the mammalian skeletal RyR
transmembrane region. The R group could be contributed either by
another protein cysteine if a disulfide is formed or could be a N if
-abS is nitrosylated. Cardiac RyRs contain stable
nitrosothiols if isolated in the absence of DTT, and nitrosylated
thiols can be denitrosylated by 10 mM DTT (Xu et al., 1998). The pig SR
used in the present experiments was isolated in the presence of 2 mM
DTT, which may not have denitrosylated all nitrosothiols on the RyRs.
Denitrosylation, however, is difficult to reconcile with the
reversibility of the effects of DTT by DTNB, unless it is also
suggested that modification per se depresses channel activity,
independent of whether the thiol is nitrosylated or oxidized by DTNB.
It seems more likely that the modified thiol is part of an intraprotein
disulfide bridge.
Two cysteine residues in the skeletal RyR, one just outside the M1
region and one between M5 and M6, are not present in cardiac RyRs (Otsu
et al., 1990). These cysteines are likely to be in the transmembrane
region and could form disulfide bridges or be available for
S-nitrosylation.
"Control-like" channel activity with 1 mM DTNB and 10 mM DTT
The observation that channel activity returned to "control-like" levels when 1 mM DTNB and 10 mM DTT were present, either on the same or opposite sides of the bilayer, provided further evidence that -aSH and -abS-R- are located in the transmembrane domain. The fact that removal of DTT, leaving only DTNB in one chamber, resulted in channel activation, which was similar to that seen when DTNB was added alone, supported the suggestion that channel activity was "control-like" when the two reagents were present together. The result suggests that redox cycling with 1 mM DTNB plus 10 mM DTT kept -abS-R- mostly in its modified form, and -aSH mostly in its reduced form.Effect of ethanol on the RyRs from pig muscle
Activation of normal RyRs and RyRMH by 1% ethanol after 2-3 min was unexpected because exposure to ethanol for the same period does not alter single-channel activity of cardiac (Eager et al., 1997) or rabbit skeletal (Ahern et al., 1997b) RyRs. However, open
times increase if rabbit skeletal RyRs are exposed to ethanol for 5 min
(Dulhunty and Curtis, unpublished observations). The results may
reflect intrinsic differences between pig and rabbit skeletal RyRs
reported previously. The maximum rate of polylysine-induced Ca2+ release is four times greater in pig skeletal SR than
that in rabbit SR (El-Hayek et al., 1995; Cifuentes et al., 1989). In addition, pig RyRs are less sensitive than rabbit RyRs to activation by
peptides corresponding to the loop between membrane-spanning segments
II and III of the skeletal DHPR (Gallant, Pace, and Dulhunty, 1999).
These functional differences are likely to be imposed by sequence
differences between the pig and rabbit RyRs. There are 45 residues (two
cysteines) that differ between the pig and rabbit RyRs in the first
1500 residues of the protein, with an overall 3% dissimilarity (Fujii
et al., 1991).
Effect of the RyR mutation in malignant hyperthermia
RyRMH responded to oxidation and reduction in a way similar to that of normal RyRs. Therefore the additional cysteine residue in MH does not alter the response of the RyR to either oxidizing or reducing reagents. Either Cis615 is buried in the protein and is not accessible to the redox reagents, or oxidation/reduction of the additional -SH or -S-R- does not effect channel activity under the conditions of our experiments. The response of RyRs to oxidation can depend on ligands bound to the protein (Eager et al., 1998; Xu et al., 1998); thus it remains possible that
Cis615 in RyRMH becomes available for oxidation
or reduction, or that the modified residue is able to regulate
activity, during increased Ca2+ release and increased
metabolic activity, and can thus further enhance Ca2+
release from the SR during the MH response.
In conclusion, the similar effects of oxidizing reagents in cardiac and
skeletal RyRs suggest that at least two of the cysteine residues whose
modification alter channel gating are conserved between the two
proteins. The results further suggest that free -SH residues and
modified thiols are present in the transmembrane region of the skeletal
RyR under control conditions and that their covalent modification under
oxidizing or reducing conditions can significantly modify RyR channel gating.
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ACKNOWLEDGMENTS |
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The authors are grateful to Suzi Pace and Joan Stivala for their
assistance in the preparation of SR vesicles from normal and MH pigs.
The isolation of the vesicles, caffeine/halthane testing, and genetic
testing were done in collaboration with Drs. Virginia Owen, Pauline
Junankar, Derek Laver, Graham Lamb, Nicole Taske, and Paul Foster
(Laver et al., 1997).
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FOOTNOTES |
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Received for publication 10 May 1999 and in final form 19 August 1999.
Address reprint requests to Dr. A. F. Dulhunty, Division of Biochemistry and Molecular Biolgy, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. Tel.: 61-2-6249-4491; Fax: 61-2-6249-4761; E-mail: angela.dulhunty{at}anu.edu.au.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Biophys J, December 1999, p. 3010-3022, Vol. 77, No. 6
© 1999 by the Biophysical Society 0006-3495/99/12/3010/13 $2.00
This article has been cited by other articles:
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, AIII and BIII only).
