ARCHIVES
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 522-528, 1992
Rose Bengal Activates the Ca*+ Release Channel from Skeletal Muscle Sarcoplasmic Reticulum’ Hui Xiong,* Edmond Buck,* Janice Stuart,ts2 Isaac N. Pessah,$ Guy Salama,$ and Jonathan J. Abramson*s3 *Departments of Physics and tchemistry, Environmental Sciences and Resources Program, Portland State University, Portland, Oregon 97207; SDepartment of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, California 95616; and $Department of Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received July 24, 1991, and in revised form October 3, 1991
The photooxidizing xanthene dye rose bengal (10 nM to 1 PM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2’ o 1992 Aoademic channel protein in the Ca2+ release process. Press,
Inc.
i This work was supported by grants from the American Heart Association (87-915), the American Cancer Society (CH-445), and NIH (lR15GM44337-01) to J.J.A.; by American Heart Association Grant (87.1065), and the Western Pennsylvania Heart Association to G.S.; and by NIH (ES05002) to I.N.P. This is Environmental Sciences and Resources Publication 264. ’ Present address: Department of Pharmacology, Oregon Health Sciences University, Portland, OR 97201. ‘To whom correspondence should be addressed at Department of Physics, Portland State University, P.O. Box 751, Portland, OR 97207. Fax: (503) 725-4882.
Abrupt reperfusion and reoxygenation of ischemic tissue results in a burst in free radical production [i.e., superoxide (OF), hydroxyl radicals (‘OH-), and singlet oxygen (lo,)]. These intermediates have been implicated in a number of aspects of cellular injury. The sarcoplasmic reticulum (SR)4 and the sarcolemma appear to be especially sensitive to reactive oxygen species (ROS) (l-4). The site(s) of action for ROS damage is unknown, although previous studies on the SR have focused on damage to the Ca2’ pump. In this paper, we demonstrate that photooxidation of rose bengal, a potent stimulator of ROS, dramatically alters the gating characteristics of the reconstituted Ca2+ release channel from skeletal muscle SR, whereas SR potassium channels are shown not to be affected. The functional changes that occur to the SR Ca2+ release process as a result of photooxidation are characterized. MATERIALS
AND
METHODS
Preparation of SR vesicles and the 106kDaprotein.
Rabbit skeletal muscle sarcoplasmic reticulum vesicles were prepared according to the method of MacLennan (5). Vesicles were further fractionated into heavy sarcoplasmic reticulum and light sarcoplasmic reticulum on a discontinuous sucrose gradient (6). [3H]Ryanodine binding assays were carried out with terminal cisternae SR vesicles, prepared according to Inui et al. (7). PDP-biotin hydrazide labeling of the 106.kDa sulfbydryl-activated Ca*+ channel protein followed the protocol of Zaidi et al. (8). Isolation of the 106.kDa protein followed this procedure with the exception that streptavidin conjugated to agarose was purchased from Molecular Probes (Eugene, OR), not immobilized on Affi-Gel 10 beads. As previously described (8), following purification of the 106-kDa channel protein, no high-molecular-weight protein (-450 kDa) was visualized on overloaded
4 Abbreviations used: SR, sarcoplasmic reticulum; Hepes, 4-(2.hydroxyethyl)-1-piperazineethanesulfonic acid; Chaps, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate; EGTA, ethylene glycol bis (P-aminoethyl ether)NN’-tetraacetic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; SPDP, N-succinimidyl 3-(2-pyridyl)dithioproponate; Car, free Cax’ concentration; AMP-PCP, P,rmethyleneadenosine 5’.triphosphate; ROS, reactive oxygen species; BLM, bilayer lipid membrane; RR, ruthenium red.
522 All
0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
Ca2+ RELEASE
FROM
SKELETAL
MUSCLE
Ag+-stained gels. SR vesicles amd the isolated 106-kDa protein were stored in liquid N, until used. Reconstitution was carried out Reconstitution into a lipid &layer. by fusion of SR vesicles (5-10 p.g) added to the cis chamber of a planar bilayer lipid membrane (BLM’), or following addition of the purified 106-kDa protein (-80 ng) to both sides of a BLM. Bilayers were made with either a 5:3 or a 1:l mixture of phosphatidylethanolamine and phosphatidylserine at 50 mg/ml in decane. In fusion experiments the cis chamber contained 500 mM CsCl, 0.5-0.7 mM CaCl,, 10 mM Hepes, pH 7.2 or 7.4, while the trans chamber contained 100 mM CsCl, 10 mM Hepes, pH 7.2 or 7.4. Following fusion, 1.5 mM EGTA, pH 7.2, was added to the cis chamber, and the cis chamber was then perfused with an identical buffer with no added Ca2+ or EGTA. In those experiments in which rose bengal-treated protein was exposed to light, the bilayer was illuminated with a halogen incandescent lamp of -8000 lux measured at the sample. All voltages were measured with respect to the tram (ground) side. K+ channel activity was monitored with a KC1 gradient (500 mM KC1 cis vs 100 mM KC1 trans) at positive voltages (lo40 mV). A List patch clamp amplifier (Model L/M-EPC7) was used to monitor picoampere currents. Data were filtered at 3 kHz, processed with a VR10 digital data recorder (Instrutech), stored on VCR tape, and subsequently analyzed for channel activity using the program pCLAMP (Axon Instruments, Burlingame, CA). Bilayer experiments were repeated as indicated at the end of each figure caption (e.g., R = 5). [3H]Ryanodine binding dissociation studies. Junctional membranes (30 pg/ml protein) were incubated at 37°C for 3 h in the presence of 1 nM [3H]ryanodine in buffer containing 20 mM Hepes, pH 7.1, 250 mM KCl, 15 mM NaCl, and 50 FM CaCl,. Samples were then exposed to either 50 or 500 nM rose bengal either in the presence of white light (6500 lux) or in near complete darkness for various amounts of time. The assays were quenched as previously described (9). SR vesicles were passively loaded by inCu’+ flux measurements. cubation overnight at 0°C in a medium containing 100 mM KCl, 5 mM MgClx, 1 mM %aClx, 50 mM Hepes, pH 7.0, at a protein concentration of 10 mg/ml. The vesicles werle then diluted 50-fold into a buffer containing 100 mM KCl, 5 mM MgCl,, 50 mM Hepes, pH 7.0, with various additions of Ca2+ and EGTA. The final free Ca*+ concentration was calculated using the stability constants reported by Fabiato and Fabiato (10). Samples were irradiated with a 360-W broad-spectrum light source (intensity of -10,000 lux measured at the sample). A beaker of water was placed between the light source and the sample to screen out infrared light. In semidark experiments, using rose bengal, the room was lit with diffuse light from an adjoining room. As a function of time, aliquots were filtered through a 0.45-Km millipore filter, and were then washed with 1 ml of an identical buffer containing 5 mM CaClz, and 5 mM MgCl,. The filter was then added to solvent free liquid scintillation fluid (Isolab, Akron, OH) and counted. The Ca*+ efflux rate was calculated from the slope of the ‘%a’+ remaining in the vesicles as a function of time. o. Materials. All materials wfere purchased from Sigma Chemical (St. Louis, MO) except for the following: dithiothreitol and Hepes bu ii er were purchased from Research Organics (Cincinnati, OH); imidazple was purchased for Aldrich Chemical Co. [3H]Ryanodine was purchaked from New England Nuclear. ‘%a was purchased from ICN (Irvine, CA). Ryanodine-dehydroryanodine was purchased from Agrisystems Int. (Wind Gap, PA).
RESULTS Fusion of SR vesicles to a planar bilayer lipid membrane has been used to examin’s activity of potassium (ll), chloride (12), and Ca2+ channels (13). In the presence of an asymmetric 5:l CsCl gradient (asdescribed under Materials and Methods) and 5 PM CaCl, (cis side), Cs’ transport through the Ca2+ release channel and channel gating were examined (Fig. 1A). Addition of 500 nM rose bengal
SARCOPLASMIC
523
RETICULUM
lo
ms
1
30 PA
1
lloldlng
Potcrlllal
(mV)
FIG. 1. Rose bengal plus light activates the Ca2+ channel but does not effect its selectivity. Single channel current fluctuations in a 5:l CsCl gradient after fusion of SR vesicles to a BLM (as described under Materials and Methods). (A) In the presence of 5 /*M cis Ca2+ (0.1 mM CaCl,, 0.1 mM EGTA), P, = 0.29. (B) 500 nM rose bengal was added to both chambers, and the light (-8000 lux) was turned on to activate channel activity, P,, = 0.88. (C) Addition of 10 PM ruthenium red. A holding potential of +20 mV with respect to the trans chamber is maintained across the BLM. (D) A single channel current-voltage curve before (O), and after (0) light activation. The single channel conductance y = 475 ps (n = 5).
in the presence of bright light increased the channel open probability (Fig. 1B). At submicromolar concentration of rose bengal, light is necessary to activate this channel. Although channel activity was stimulated, the conductance (475 pS), and the selectivity of the channel remained unaffected by photoactivation of rose bengal (Fig. 1D). The channel remained highly cation selective (Pc,+/Pclr = 13; Fig. lD), and sensitive to inhibition by 10 PM ruthenium red (RR; Fig. 1C). The relative abundance of chloride channels was found to vary from one preparation of SR to the next. The voltage intercept at zero current is related to the relative cation versus anion permeability of the membrane. Activation of the Ca2+ channel by rose bengal resulted in no corresponding modification of the background current levels or the corresponding voltage intercept at zero current. This indicated that there had been no preferential activation of SR Cl- or K+ channels by rose bengal.
524
XIONG
Potassium channels from SR vesicles have been extensively studied by Miller and co-workers (ll), using the vesicle fusion technique. In Fig. 2, in the presence of a 5: 1 KC1 gradient, and a low concentration of cis Ca2’ (Caf - 6 nM inactivates the SR Ca2+ channel), a K+ channel was observed. Addition of 500 nM rose bengal, in the dark, had no effect on these relatively slow fluctuations (Fig. 2B). However, upon addition of light, a high conductance, rapid fluctuation was observed (Fig. 2C). The conductance of this channel (-1000 pS) is similar to that reported by Smith et al. for the K+ conductance of the Ca2+ release protein in a symmetric 0.5 M KC1 solution (15). Identification of these fluctuations as the Ca2+ channel was further confirmed by the observation that the addition of 5 PM RR inhibits these rapid large fluctuations (Fig. 2D). Subsequent to the addition of RR, the slow, lower conductance K+ channel was once again observed. The 170pS K+ channel was inhibited (75%) by 50 mM CsCl (data not shown), as previously demonstrated (16). No stimulation of K+ channels by rose bengal and light was observed prior to the activation of the Ca2’ channel by photooxidation. Furthermore, the low free Ca2+ concentration did not prevent photoactivation of the K’ conducting Ca2+ release protein in the presence of rose bengal.
B
D
30ms 1 30pA
FIG. 2. Activation of SR Gas+ channels by rose bengal does not effect K+ channels. SR fusion to a BLM was carried out in the presence of a fivefold KC1 gradient (500 mM KCl, 5 mM Hepes, pH 7.2, in the cis chamber; 100 mM KU, 5 mM Hepes, pH 7.2, in the trans chamber) as described under Materials and Methods. The free Ca2+ concentration in the cis chamber was -6 nM. (A) Control Kf channels. (B) Addition of 500 nM rose bengal in the dark. (C) Following exposure to light (8000 lux for 15 s), and (D) addition of 5 pM ruthenium red to the cis chamber. The holding potential was +25 mV with respect to the tramsside of the BLM (n = 3).
5-
1
/
1 8
/
6
I
I
8
4
PC0
FIG. 3. Ca*+ dependence of rose bengal-induced Ca’+ release from skeletal muscle SR vesicles. SR vesicles were passively loaded with 46CaC12as described under Materials and Methods at a high Mgs+ concentration (5 mM). They were then diluted into solutions buffered with EGTA-Ca that yield the pCa shown on the abscissa. The samples were then irradiated with a 360-W light source in the presence (m) or absence (0) of 200 nM rose bengal. Aliquots were collected as a function of time, filtered, and washed as described under Materials and Methods. The calculated Ca*+ elllux rate is plotted on the ordinate.
Figure 3 shows that rose bengal-stimulated Ca2+ release from passively loaded SR vesicles was also insensitive to the free Ca2+ concentration. The low release rates and absence of a measurable Ca2’ dependence in the control for Fig. 3 were due to the high Mg2+ concentration (5 mM) used in these experiments. Following activation by rose bengal, no Ca2+ dependence of the release rate was observed. This is in contrast to other methods of stimulating Ca2+ release in the presence of millimolar concentrations of Mg2’ which showed a marked activation by micromolar concentrations of Ca2’ (24). Not only was the Ca2+ release rate stimulated by photooxidation of rose bengal, but as shown in Fig. 2, at low free Ca2+ concentrations (6 nM), Ca2+ channel activity was also activated. In the absence of direct light, no activation of channel activity was observed. As shown in Fig. 4, activation of the Ca2+ release protein by 1 PM rose bengal and exposure to light was independent of the side to which the dye was added. It has previously been shown that addition of micromolar concentrations of ryanodine to JSR vesicles following fusion to a BLM results in locking of the Ca2’ release channel in a half conductance state (18). As shown in Fig. 5, addition of 0.7 pM ryanodine to the cis chamber results in a slowly fluctuating channel of approximately 60% full conductance. Subsequent addition of 0.6 pM rose bengal to the cis chamber, followed by introduction of light, reverses the effect of ryanodine (Fig. 5B). The channel returns to a rapidly fluctuating full conductance state.
Ca?’ RELEASE
FROM
SKELETAL
MUSCLE
SARCOPLASMIC
525
RETICULUM
40 ms 1
20 ms
1
30 PA
FIG. 4. Activation of channel activity by rose bengal is side independent. Following fusion of SR vesicles to a PE:PS (1:l) BLM, Ca-EGTA was buffered in the cis chamber to yield a free Ca2+ concentration of 160 nM. (A) Control trace with a 5:l CsCl gradient as described in the legend to Fig. 1. (B) Following addition of 1 yM rose bengal to the trans chamber and activation by light (n = 3). (C) Repeat with a new BLM, same conditions as in (A). (D) Following addition of 1 pM rose bengal to the cis chamber and activation by light. The holding potential is +20 mV (n = 6).
It was shown in the previous paper that nanomolar concentrations of rose bengal inhibit high affinity [3H]ryanodine binding tot its receptor in a light-dependent manner (19). The reversal of the slowly fluctuating ryanodine-stimulated subconductance state induced by the addition of rose bengal (Fig. 5) implies that rose bengal can displace bound ryanodine from its receptor. This was confirmed in Fig. 6, in wlhich [3H]ryanodine bound to the high affinity site was directly shown to be rapidly displaced by addition of rose blengal. Displacement of bound [3H]ryanodine was dependent on the rose bengal concentration and requires the presence of light. We have previously identified and isolated a sulfhydrylactivated channel protein, which when reconstituted into a planar BLM showed characteristics in common with those expected of the Ca2+ release protein from skeletal muscle SR (8). The activity of this protein of molecular mass 106 kDa was stimulated by Ca’+, ATP, doxorubicin, and Agf, and was inhibited by Mgzf and ruthenium red. The activity of the reconstituted 106-kDa protein was also stimulated by addition of 500 nM rose bengal (Fig. 7B). Channel activity was further increased by the addition of light (Fig. 7C). If 10 nM ryanodine was added to the reconstituted 106kDa protein before the (addition of rose bengal (Figs. 8C
FIG. 5. Ryanodine modification of the Ca2+ release channel is reversed by rose bengal. Following the protocol described in the legend to Fig. 4, with the free Ca2+ concentration buffered at 100 pM in trace (A), 0.7 pM ryanodine was added to the cis chamber. (B) Following addition of 600 nM rose bengal and activation by light. The holding potential was +20 mV (n = 3).
and 8D), channel activity was modified to a long subconductance state (with infrequent closures to the ground shown). Subsequent addition state -1 per second-not of 500 nM rose bengal, plus exposure to light, reactivated the channel to a rapidly fluctuating full conductance state (Figs. 8E and 8F), similar to what was observed in Fig. 5, following fusion of SR vesicles to a bilayer. Although the
0
1
2
3
4
5
10
Time (min) FIG. 6. Displacement of [3H]ryanodine from high affinity receptor sites by rose bengal is light dependent. Light-dependent dissociation of the [3H]ryanodine receptor equilibrium complex by 50 nM (V) and 500 nM (0) rose bengal. Junctional SR membranes (30 yg) were equilibrated with 1 nM [3H]ryanodine for 3 h. Rose bengal was added at subsequent times as indicated in near complete darkness (0, v) or in the presence of white light of -6500 lux (0, V). The mean maximum specific binding in the controls lacking rose bengal averaged 5163 f 385 dpm. The data presented are from a single determination performed in duplicate, which was repeated once with essentially the same results.
526
XIONG
90 ms 20~A
1
FIG. 7. Light-dependent stimulation of the 106-kDa channel protein by rose bengal. Following addition of -80 rig/ml of the isolated 106kDa protein to both sides of a BLM [PE:PS:PC (5:3:2) at 50 mg/ml in decane] in a symmetric NaCl buffer (250 mM), 100 pM Ca2+ was added to both sides of the BLM (A). In trace (A), the open probability, P, = 0.09. In trace (B), 500 nM rose bengal was added to both sides of the BLM (P, = 0.20). (C) Following addition of light (P, = 0.71). The holding potential for all traces equals +20 mV (n = 3).
voltage dependence of channel gating is not very dramatic, it should also be noted that the probability of finding the channel in an open state is larger at positive potentials than at negative potentials, following reconstitution of the isolated 106-kDa protein (Fig. 8A vs 8B; 8E vs 8F). In other reconstitution experiments, carried out with the isolated 106-kDa protein, a voltage asymmetry was always evident. However, depending on the orientation of the protein upon incorporation, in some experiments the channel activity was larger at more negative potentials. This asymmetric voltage dependence was present either before or after activation by rose bengal. It has also previously been reported following fusion of SR vesicles to a BLM (20). The single channel conductance in the presence of symmetric solutions of 250 mM NaCl, following fusion of SR vesicles to a bilayer (y = 400 pS; not shown) is the same as observed following incorporation of the isolated 106-kDa protein into the BLM (Figs. 7A, 8A).
ET AL.
H,O is between 2 and 5 pus,and is considerably longer in organic solvents (21). Given the high diffusion coefficient of O2 in water (D = lo-’ m2 s-l), it is likely that ‘02 diffuses at least 10 times the thickness of the bilayer before decaying. It is therefore not surprising that photoactivation of the release channel is independent of the side to which the rose bengal is added (Fig. 4). The Ca2+ release protein from skeletal muscle sarcoplasmic reticulum has previously been shown to be highly cation selective (13). However, the channel only slightly distinguishes between monovalent and divalent cations (15). The use of a high concentration CsCl gradient simplifies our analysis. Under these conditions, the SR K’ channel was strongly inhibited (16). At positive membrane potentials, near the reversal potential for chloride, Clfluxes were relatively insignificant, and we were then able to focus on Cs+ transport through the Ca2+ release channel. The Cs+ conducting Ca2+ release channel was inhibited by ruthenium red, and was activated by rose bengal
F
90 ms
DISCUSSION
Exposure of the photooxidizing dye rose bengal to light results in the production of ‘02, and the rapid release of Ca2+ from skeletal muscle SR vesicles (19). In this paper we show that the Ca2+ release channel from SR is activated by rose bengal in the presence of light (P,, increases), but the single channel conductance is unaffected. The nature of the activation is independent of the side to which rose bengal is added. The lifetime of singlet oxygen in
1 IOPA FIG. 8. Ryanodine-induced subconductance state is reversed by rose bengal. The 106-kDa protein was reconstituted into a BLM as described in the legend to Fig. 7. (A) Control trace at +40 mV, P, = 0.99. (B) Control trace at -40 mV, P, = 0.92. (C) Following addition of 10 nM ryanodine to both chambers at +40 mV, and (D) at -40 mV. (E) Addition of 500 nM rose bengal to both sides of the BLM in the presence of light. Recorded at +40 mV. P, = 0.93. (F) Same as (E) at -40 mV. P, = 0.12 (72 = 3).
Ca2+ RELEASE
FROM
SKELETAL
MUSCLE
in the light (Fig. 1). The rose bengal-activated channel did not show spontaneous inactivation. Following light exposure for 15 to 30 s, the channel was stable in the dark for periods of greater than 30 min. Neither the cation selectivity of the Ca2’ release channel (Fig. 1) nor the selectivity of the background current was modified by rose bengal. This latter observation indicated that no additional transport systems with different cation versus anion selectivity (i.e., K+ or Cl- channels) were being activated. This was confirmed in Fig. 2, in which K+ channels were shown to be unaffected by rose bengal and light. Activation of the relea.se channel from SR by adenine nucleotides (22,23), ryanodine (18), and doxorubicin (2426) has been shown to be Ca2+ dependent. Maximal activation occurs between 1 and 10 pM Ca2+. At Ca2+ concentrations as low as 10 nM the channel has been shown to be totally inhibited (22). Rose bengal-induced Ca2+ release and rose bengal activation of the Ca2’ release channel are both observed to be insensitive to Ca” (Figs. 2, 3). It is likely that the molecular mechanism underlying rose bengal-induced Ca” release is different than previously observed with other activating reagents. As shown in the previous paper (1!3), rose bengal-induced Cazf release is insensitive to Mg’+, while other methods of stimulating Ca2+ release are inhibited by millimolar concentrations of Mg2+ (22-24, 27-29). Furthermore, ATP has been shown to stimulate Ca2+ release from SR vesicles and to stimulate the Cazt release channel (22, 23). In the previous study, AMP-PCP, a nonhydrolyzable analog of ATP was shown to inhibit rose bengal-induced Ca2+ release from SR vesicles (19). In spite of this apparent difference in the mechanism of interaction with the -release channel, it is clear from this and the previous study that rose bengal interacts directly with the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum. Ryanodine’s ability to lock the release channel in a subconductance state (Fig. 5) was reversed upon addition of rose bengal, as was high affinity bound [3H]ryanodine (Fig. 6). Also, both the fusion studies with SR vesicles and the reconstitution experiments with the isolated 106-kDa sulfhydryl-activated protein indicate that rose bengal directly activated the Ca2+ release channel protein from SR. In comparing the properties of SR vesicles following fusion to a BLM and those of the reconstituted isolated 106-kDa protein most characteristics are similar. In both types of experiments, we observed a stimulation of channel activity upon addition of rose bengal and introduction of light (Figs. 1,7). In both cases, rose bengal was observed to reverse ryanodine modification of the Ca2+ release channel (Figs. 5, 8). Also, the conductance of the release channel in both the isolated 106-kDa protein preparation and the Ca2+ release channel following fusion of a SR vesicle to a BLM was identical. The differences between the two types of reconstitution experiments were more subtle. The sensitivity of the 106kDa protein to both rose bengal and ryanodine was greater
SARCOPLASMIC
RETICULUM
527
than that of the BLM following fusion of the SR. Where 10 nM ryanodine locked the 106-kDa protein in a subconductance state (Fig. 8), higher concentrations were needed to produce a similar effect following fusion to a BLM (Fig. 5). A concentration of 10 nM ryanodine slowed the gating of the release channel following fusion, but did not modify its conductance following fusion (30, 31). Concentrations of ryanodine slightly less than 1 pM also caused the channel following fusion to show subconductante states (Fig. 5). In a previous study, we observed that a minimum of 100 nM ryanodine was required in order to observe a subconductance state following fusion of SR vesicles to a BLM (31). The only experiments in which we noted a stimulation of channel activity by 500 nM rose bengal in the dark were carried out with the reconstituted 106-kDa protein (Fig. 7). In this experiment, we also observed a further activation by the addition of light. At the same rose bengal concentration, a stimulation of channel activity was not observed in the dark in the experiments carried out following fusion of SR vesicles to a BLM. In all SR fusion experiments in which rose bengal (500 nm1 pM) was added to either side of the BLM, activation of channel activity was observed within 20-30 s following exposure to light. Aside from the greater sensitivity of the reconstituted 106-kDa protein to ryanodine and rose bengal, the properties of the isolated protein and the SR following fusion to the BLM were strikingly similar. There was a strong correlation between the properties of the isolated 106kDa protein and the characteristics of rose bengal-stimulated Ca2+ release from SR vesicles. The site of action of photoinduced modification to the SR appears to be the 106-kDa sulfhydryl-activated Ca2+ channel protein. The fact that the characteristics of the 106-kDa protein and the SR following fusion agree so well gives strong credence to the identification of the 106-kDa protein as the Ca2+ release protein from sarcoplasmic reticulum. The sarcoplasmic reticulum has previously been implicated as a site of action for reactive oxygen damage. However, previous studies have focused on the Ca’+, Mg2+-ATPase as the primary target. This and the previous paper demonstrate that the Ca2+ release pathway is more sensitive to ROS damage than is the Ca2+ pump. Moreover, rapid Ca2+ release is induced by nanomolar concentrations of rose bengal in the presence of light by directly interacting with the ryanodine receptor, Ca2+ release protein, 106-kDa sulfhydryl-activated channel protein. A recent abstract by Cumming et ~2. (32) indicates that sheep cardiac SR shows a sensitivity to rose bengal similar to that of skeletal muscle SR. Whether or not the cardiac SR Ca2+ release protein is the primary site of action for ROS damage during reperfusion of cardiac muscle will require further detailed studies. REFERENCES 1. Manning, 497-518.
A. S., and Hearse, D. J. (1984) J. Mol. Cell. Cardiol.
16,
528
XIONG
2. Hess, M. L., and Manson, N. H. (1984) J. Mol. Cell. Cardiol. 16, 969-985. 3. Kukreja, R. C., Okabe, E., Schrier, G. M., and Hess, M. L. (1988) Arch. Biochem. Biophys. 261,441-457. 4. Kim, M-S., and Akera, T. (1987) Am. J. Physiol. 252, H252-H257. 5. MacLennan, D. H. (1970) J. Biol. Chem. 245,4508-4518. 6. Salama, G., and Abramson, J. J. (1984) J. Biol. Chem. 259, 13,36313,369.
7. Inui, M., Saito, A., Fleischer, S. (1987) J. Biol. Chem. 262, 17401747.
8. Zaidi, N. F., Lagenaur, C. F., Hilkert, 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
R. J., Xiong, H., Abramson, J. J., and Salama, G. (1989) J. Biol. Chem. 264, 21,737-21,747. Pessah, I. N., Stambuk, R. A., and Casida, J. E. (1987) Mol. Phurmacol. 31,232-238. Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505. Miller, C., Bell, J. E., and Garcia, A. M. (1984) in Current Topics in Membranes and Transport (Stein, W. D., Ed., Vol. 21, pp. 99132, Academic Press, New York. Rousseau, E. (1989) J. Membr. Biol. 110, 39-47. Smith, J. S., Coronado, R., and Meissner, G. (1985) Nature 316, 446-449. Ohnishi, S. T. (1979) J. Biochem. 86, 1147-1150. Smith, J. S., Imagawa, T., Ma, J., Fill, M., Campbell, K. P., and Coronado, R. (1988) J. Gen. Physiol. 92, l-26. Coronado, R., and Miller, C. (1979) Nature 280,807-810. Smith, J. S., Coronado, R., and Meissner, G. (1986) Biophys. J. 50, 921-928. Rousseau, E., Smith, J. S., and Meissner, G. (1987) Am. J. Physiol.
253,C364-C368.
ET AL. 19. Stuart, J., Pessah, I. N., Favero, T. G., and Abramson, Arch. Biochem. Biophys. 292, 512-521.
J. J. (1991)
20. Ma, J., Fill, M., Knudson, C. M., Campbell, K. P., and Coronado, R. (1988) Science 242, 99-102.
21. Perez, H. D. (1985) in Handbook Research (Greenwald, Raton, FL.
of Methods for Oxygen Radical R. A., Ed.), pp. 111-113, CRC Press, Boca
22. Smith, J. S., Coronado, R., and Meissner, G. (1986) J. Gen. Physiol. 88,573-588. 23. Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374. 24. Abramson, J. J., Buck, E., Salama, G., Casida, J. E., and Pessah, I. N. (1988) J. Biol. Chem. 263, l&750-18,758.
25. Holmberg, S. R. M., and Williams, A. J. (1990) Circ. Res. 67, 272283. 26. Ondrias, K., Borgatta, L., Kim, D. H., and Ehrlich, B. E. (1990) Circ. Res. 67, 1167-1174.
27. Salama, G., and Abramson, J. (1984) J. Biol. Chem. 259, 13,36313,369.
28. Trimm, J. L., Salama, G., and Abramson, J. J. (1986) J. Biol. Chem. 261, 16,092-16,098. 29. Abramson, J. J., Cronin, J., and Salama, G. (1988) Arch. Biochem. Biophys. 263, 245-255.
30. Bull, R., Marengo,
J. J., Suarez-Isla, B. A., Donoso, J. L., and Hidalgo, C. (1989) Biophys. J. 56, 749-756.
31. Zimanyi, Submitted
I., Buck, E., Abramson, for publication.
32. Cumming, D., Holmberg,
P., Sutko,
J. J., and Pessah, I. N. (1991)
S., Kusama, Y., Shattock, liams, A. (1990) J. Physiol. 420, 88P.
M., and Wil-
OF BIOCHEMISTRY
Vol. 292, No. 2, February
AND
BIOPHYSICS
1, pp. 522-528, 1992
Rose Bengal Activates the Ca*+ Release Channel from Skeletal Muscle Sarcoplasmic Reticulum’ Hui Xiong,* Edmond Buck,* Janice Stuart,ts2 Isaac N. Pessah,$ Guy Salama,$ and Jonathan J. Abramson*s3 *Departments of Physics and tchemistry, Environmental Sciences and Resources Program, Portland State University, Portland, Oregon 97207; SDepartment of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, California 95616; and $Department of Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received July 24, 1991, and in revised form October 3, 1991
The photooxidizing xanthene dye rose bengal (10 nM to 1 PM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2’ o 1992 Aoademic channel protein in the Ca2+ release process. Press,
Inc.
i This work was supported by grants from the American Heart Association (87-915), the American Cancer Society (CH-445), and NIH (lR15GM44337-01) to J.J.A.; by American Heart Association Grant (87.1065), and the Western Pennsylvania Heart Association to G.S.; and by NIH (ES05002) to I.N.P. This is Environmental Sciences and Resources Publication 264. ’ Present address: Department of Pharmacology, Oregon Health Sciences University, Portland, OR 97201. ‘To whom correspondence should be addressed at Department of Physics, Portland State University, P.O. Box 751, Portland, OR 97207. Fax: (503) 725-4882.
Abrupt reperfusion and reoxygenation of ischemic tissue results in a burst in free radical production [i.e., superoxide (OF), hydroxyl radicals (‘OH-), and singlet oxygen (lo,)]. These intermediates have been implicated in a number of aspects of cellular injury. The sarcoplasmic reticulum (SR)4 and the sarcolemma appear to be especially sensitive to reactive oxygen species (ROS) (l-4). The site(s) of action for ROS damage is unknown, although previous studies on the SR have focused on damage to the Ca2’ pump. In this paper, we demonstrate that photooxidation of rose bengal, a potent stimulator of ROS, dramatically alters the gating characteristics of the reconstituted Ca2+ release channel from skeletal muscle SR, whereas SR potassium channels are shown not to be affected. The functional changes that occur to the SR Ca2+ release process as a result of photooxidation are characterized. MATERIALS
AND
METHODS
Preparation of SR vesicles and the 106kDaprotein.
Rabbit skeletal muscle sarcoplasmic reticulum vesicles were prepared according to the method of MacLennan (5). Vesicles were further fractionated into heavy sarcoplasmic reticulum and light sarcoplasmic reticulum on a discontinuous sucrose gradient (6). [3H]Ryanodine binding assays were carried out with terminal cisternae SR vesicles, prepared according to Inui et al. (7). PDP-biotin hydrazide labeling of the 106.kDa sulfbydryl-activated Ca*+ channel protein followed the protocol of Zaidi et al. (8). Isolation of the 106.kDa protein followed this procedure with the exception that streptavidin conjugated to agarose was purchased from Molecular Probes (Eugene, OR), not immobilized on Affi-Gel 10 beads. As previously described (8), following purification of the 106-kDa channel protein, no high-molecular-weight protein (-450 kDa) was visualized on overloaded
4 Abbreviations used: SR, sarcoplasmic reticulum; Hepes, 4-(2.hydroxyethyl)-1-piperazineethanesulfonic acid; Chaps, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate; EGTA, ethylene glycol bis (P-aminoethyl ether)NN’-tetraacetic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; SPDP, N-succinimidyl 3-(2-pyridyl)dithioproponate; Car, free Cax’ concentration; AMP-PCP, P,rmethyleneadenosine 5’.triphosphate; ROS, reactive oxygen species; BLM, bilayer lipid membrane; RR, ruthenium red.
522 All
0003.9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
Ca2+ RELEASE
FROM
SKELETAL
MUSCLE
Ag+-stained gels. SR vesicles amd the isolated 106-kDa protein were stored in liquid N, until used. Reconstitution was carried out Reconstitution into a lipid &layer. by fusion of SR vesicles (5-10 p.g) added to the cis chamber of a planar bilayer lipid membrane (BLM’), or following addition of the purified 106-kDa protein (-80 ng) to both sides of a BLM. Bilayers were made with either a 5:3 or a 1:l mixture of phosphatidylethanolamine and phosphatidylserine at 50 mg/ml in decane. In fusion experiments the cis chamber contained 500 mM CsCl, 0.5-0.7 mM CaCl,, 10 mM Hepes, pH 7.2 or 7.4, while the trans chamber contained 100 mM CsCl, 10 mM Hepes, pH 7.2 or 7.4. Following fusion, 1.5 mM EGTA, pH 7.2, was added to the cis chamber, and the cis chamber was then perfused with an identical buffer with no added Ca2+ or EGTA. In those experiments in which rose bengal-treated protein was exposed to light, the bilayer was illuminated with a halogen incandescent lamp of -8000 lux measured at the sample. All voltages were measured with respect to the tram (ground) side. K+ channel activity was monitored with a KC1 gradient (500 mM KC1 cis vs 100 mM KC1 trans) at positive voltages (lo40 mV). A List patch clamp amplifier (Model L/M-EPC7) was used to monitor picoampere currents. Data were filtered at 3 kHz, processed with a VR10 digital data recorder (Instrutech), stored on VCR tape, and subsequently analyzed for channel activity using the program pCLAMP (Axon Instruments, Burlingame, CA). Bilayer experiments were repeated as indicated at the end of each figure caption (e.g., R = 5). [3H]Ryanodine binding dissociation studies. Junctional membranes (30 pg/ml protein) were incubated at 37°C for 3 h in the presence of 1 nM [3H]ryanodine in buffer containing 20 mM Hepes, pH 7.1, 250 mM KCl, 15 mM NaCl, and 50 FM CaCl,. Samples were then exposed to either 50 or 500 nM rose bengal either in the presence of white light (6500 lux) or in near complete darkness for various amounts of time. The assays were quenched as previously described (9). SR vesicles were passively loaded by inCu’+ flux measurements. cubation overnight at 0°C in a medium containing 100 mM KCl, 5 mM MgClx, 1 mM %aClx, 50 mM Hepes, pH 7.0, at a protein concentration of 10 mg/ml. The vesicles werle then diluted 50-fold into a buffer containing 100 mM KCl, 5 mM MgCl,, 50 mM Hepes, pH 7.0, with various additions of Ca2+ and EGTA. The final free Ca*+ concentration was calculated using the stability constants reported by Fabiato and Fabiato (10). Samples were irradiated with a 360-W broad-spectrum light source (intensity of -10,000 lux measured at the sample). A beaker of water was placed between the light source and the sample to screen out infrared light. In semidark experiments, using rose bengal, the room was lit with diffuse light from an adjoining room. As a function of time, aliquots were filtered through a 0.45-Km millipore filter, and were then washed with 1 ml of an identical buffer containing 5 mM CaClz, and 5 mM MgCl,. The filter was then added to solvent free liquid scintillation fluid (Isolab, Akron, OH) and counted. The Ca*+ efflux rate was calculated from the slope of the ‘%a’+ remaining in the vesicles as a function of time. o. Materials. All materials wfere purchased from Sigma Chemical (St. Louis, MO) except for the following: dithiothreitol and Hepes bu ii er were purchased from Research Organics (Cincinnati, OH); imidazple was purchased for Aldrich Chemical Co. [3H]Ryanodine was purchaked from New England Nuclear. ‘%a was purchased from ICN (Irvine, CA). Ryanodine-dehydroryanodine was purchased from Agrisystems Int. (Wind Gap, PA).
RESULTS Fusion of SR vesicles to a planar bilayer lipid membrane has been used to examin’s activity of potassium (ll), chloride (12), and Ca2+ channels (13). In the presence of an asymmetric 5:l CsCl gradient (asdescribed under Materials and Methods) and 5 PM CaCl, (cis side), Cs’ transport through the Ca2+ release channel and channel gating were examined (Fig. 1A). Addition of 500 nM rose bengal
SARCOPLASMIC
523
RETICULUM
lo
ms
1
30 PA
1
lloldlng
Potcrlllal
(mV)
FIG. 1. Rose bengal plus light activates the Ca2+ channel but does not effect its selectivity. Single channel current fluctuations in a 5:l CsCl gradient after fusion of SR vesicles to a BLM (as described under Materials and Methods). (A) In the presence of 5 /*M cis Ca2+ (0.1 mM CaCl,, 0.1 mM EGTA), P, = 0.29. (B) 500 nM rose bengal was added to both chambers, and the light (-8000 lux) was turned on to activate channel activity, P,, = 0.88. (C) Addition of 10 PM ruthenium red. A holding potential of +20 mV with respect to the trans chamber is maintained across the BLM. (D) A single channel current-voltage curve before (O), and after (0) light activation. The single channel conductance y = 475 ps (n = 5).
in the presence of bright light increased the channel open probability (Fig. 1B). At submicromolar concentration of rose bengal, light is necessary to activate this channel. Although channel activity was stimulated, the conductance (475 pS), and the selectivity of the channel remained unaffected by photoactivation of rose bengal (Fig. 1D). The channel remained highly cation selective (Pc,+/Pclr = 13; Fig. lD), and sensitive to inhibition by 10 PM ruthenium red (RR; Fig. 1C). The relative abundance of chloride channels was found to vary from one preparation of SR to the next. The voltage intercept at zero current is related to the relative cation versus anion permeability of the membrane. Activation of the Ca2+ channel by rose bengal resulted in no corresponding modification of the background current levels or the corresponding voltage intercept at zero current. This indicated that there had been no preferential activation of SR Cl- or K+ channels by rose bengal.
524
XIONG
Potassium channels from SR vesicles have been extensively studied by Miller and co-workers (ll), using the vesicle fusion technique. In Fig. 2, in the presence of a 5: 1 KC1 gradient, and a low concentration of cis Ca2’ (Caf - 6 nM inactivates the SR Ca2+ channel), a K+ channel was observed. Addition of 500 nM rose bengal, in the dark, had no effect on these relatively slow fluctuations (Fig. 2B). However, upon addition of light, a high conductance, rapid fluctuation was observed (Fig. 2C). The conductance of this channel (-1000 pS) is similar to that reported by Smith et al. for the K+ conductance of the Ca2+ release protein in a symmetric 0.5 M KC1 solution (15). Identification of these fluctuations as the Ca2+ channel was further confirmed by the observation that the addition of 5 PM RR inhibits these rapid large fluctuations (Fig. 2D). Subsequent to the addition of RR, the slow, lower conductance K+ channel was once again observed. The 170pS K+ channel was inhibited (75%) by 50 mM CsCl (data not shown), as previously demonstrated (16). No stimulation of K+ channels by rose bengal and light was observed prior to the activation of the Ca2’ channel by photooxidation. Furthermore, the low free Ca2+ concentration did not prevent photoactivation of the K’ conducting Ca2+ release protein in the presence of rose bengal.
B
D
30ms 1 30pA
FIG. 2. Activation of SR Gas+ channels by rose bengal does not effect K+ channels. SR fusion to a BLM was carried out in the presence of a fivefold KC1 gradient (500 mM KCl, 5 mM Hepes, pH 7.2, in the cis chamber; 100 mM KU, 5 mM Hepes, pH 7.2, in the trans chamber) as described under Materials and Methods. The free Ca2+ concentration in the cis chamber was -6 nM. (A) Control Kf channels. (B) Addition of 500 nM rose bengal in the dark. (C) Following exposure to light (8000 lux for 15 s), and (D) addition of 5 pM ruthenium red to the cis chamber. The holding potential was +25 mV with respect to the tramsside of the BLM (n = 3).
5-
1
/
1 8
/
6
I
I
8
4
PC0
FIG. 3. Ca*+ dependence of rose bengal-induced Ca’+ release from skeletal muscle SR vesicles. SR vesicles were passively loaded with 46CaC12as described under Materials and Methods at a high Mgs+ concentration (5 mM). They were then diluted into solutions buffered with EGTA-Ca that yield the pCa shown on the abscissa. The samples were then irradiated with a 360-W light source in the presence (m) or absence (0) of 200 nM rose bengal. Aliquots were collected as a function of time, filtered, and washed as described under Materials and Methods. The calculated Ca*+ elllux rate is plotted on the ordinate.
Figure 3 shows that rose bengal-stimulated Ca2+ release from passively loaded SR vesicles was also insensitive to the free Ca2+ concentration. The low release rates and absence of a measurable Ca2’ dependence in the control for Fig. 3 were due to the high Mg2+ concentration (5 mM) used in these experiments. Following activation by rose bengal, no Ca2+ dependence of the release rate was observed. This is in contrast to other methods of stimulating Ca2+ release in the presence of millimolar concentrations of Mg2’ which showed a marked activation by micromolar concentrations of Ca2’ (24). Not only was the Ca2+ release rate stimulated by photooxidation of rose bengal, but as shown in Fig. 2, at low free Ca2+ concentrations (6 nM), Ca2+ channel activity was also activated. In the absence of direct light, no activation of channel activity was observed. As shown in Fig. 4, activation of the Ca2+ release protein by 1 PM rose bengal and exposure to light was independent of the side to which the dye was added. It has previously been shown that addition of micromolar concentrations of ryanodine to JSR vesicles following fusion to a BLM results in locking of the Ca2’ release channel in a half conductance state (18). As shown in Fig. 5, addition of 0.7 pM ryanodine to the cis chamber results in a slowly fluctuating channel of approximately 60% full conductance. Subsequent addition of 0.6 pM rose bengal to the cis chamber, followed by introduction of light, reverses the effect of ryanodine (Fig. 5B). The channel returns to a rapidly fluctuating full conductance state.
Ca?’ RELEASE
FROM
SKELETAL
MUSCLE
SARCOPLASMIC
525
RETICULUM
40 ms 1
20 ms
1
30 PA
FIG. 4. Activation of channel activity by rose bengal is side independent. Following fusion of SR vesicles to a PE:PS (1:l) BLM, Ca-EGTA was buffered in the cis chamber to yield a free Ca2+ concentration of 160 nM. (A) Control trace with a 5:l CsCl gradient as described in the legend to Fig. 1. (B) Following addition of 1 yM rose bengal to the trans chamber and activation by light (n = 3). (C) Repeat with a new BLM, same conditions as in (A). (D) Following addition of 1 pM rose bengal to the cis chamber and activation by light. The holding potential is +20 mV (n = 6).
It was shown in the previous paper that nanomolar concentrations of rose bengal inhibit high affinity [3H]ryanodine binding tot its receptor in a light-dependent manner (19). The reversal of the slowly fluctuating ryanodine-stimulated subconductance state induced by the addition of rose bengal (Fig. 5) implies that rose bengal can displace bound ryanodine from its receptor. This was confirmed in Fig. 6, in wlhich [3H]ryanodine bound to the high affinity site was directly shown to be rapidly displaced by addition of rose blengal. Displacement of bound [3H]ryanodine was dependent on the rose bengal concentration and requires the presence of light. We have previously identified and isolated a sulfhydrylactivated channel protein, which when reconstituted into a planar BLM showed characteristics in common with those expected of the Ca2+ release protein from skeletal muscle SR (8). The activity of this protein of molecular mass 106 kDa was stimulated by Ca’+, ATP, doxorubicin, and Agf, and was inhibited by Mgzf and ruthenium red. The activity of the reconstituted 106-kDa protein was also stimulated by addition of 500 nM rose bengal (Fig. 7B). Channel activity was further increased by the addition of light (Fig. 7C). If 10 nM ryanodine was added to the reconstituted 106kDa protein before the (addition of rose bengal (Figs. 8C
FIG. 5. Ryanodine modification of the Ca2+ release channel is reversed by rose bengal. Following the protocol described in the legend to Fig. 4, with the free Ca2+ concentration buffered at 100 pM in trace (A), 0.7 pM ryanodine was added to the cis chamber. (B) Following addition of 600 nM rose bengal and activation by light. The holding potential was +20 mV (n = 3).
and 8D), channel activity was modified to a long subconductance state (with infrequent closures to the ground shown). Subsequent addition state -1 per second-not of 500 nM rose bengal, plus exposure to light, reactivated the channel to a rapidly fluctuating full conductance state (Figs. 8E and 8F), similar to what was observed in Fig. 5, following fusion of SR vesicles to a bilayer. Although the
0
1
2
3
4
5
10
Time (min) FIG. 6. Displacement of [3H]ryanodine from high affinity receptor sites by rose bengal is light dependent. Light-dependent dissociation of the [3H]ryanodine receptor equilibrium complex by 50 nM (V) and 500 nM (0) rose bengal. Junctional SR membranes (30 yg) were equilibrated with 1 nM [3H]ryanodine for 3 h. Rose bengal was added at subsequent times as indicated in near complete darkness (0, v) or in the presence of white light of -6500 lux (0, V). The mean maximum specific binding in the controls lacking rose bengal averaged 5163 f 385 dpm. The data presented are from a single determination performed in duplicate, which was repeated once with essentially the same results.
526
XIONG
90 ms 20~A
1
FIG. 7. Light-dependent stimulation of the 106-kDa channel protein by rose bengal. Following addition of -80 rig/ml of the isolated 106kDa protein to both sides of a BLM [PE:PS:PC (5:3:2) at 50 mg/ml in decane] in a symmetric NaCl buffer (250 mM), 100 pM Ca2+ was added to both sides of the BLM (A). In trace (A), the open probability, P, = 0.09. In trace (B), 500 nM rose bengal was added to both sides of the BLM (P, = 0.20). (C) Following addition of light (P, = 0.71). The holding potential for all traces equals +20 mV (n = 3).
voltage dependence of channel gating is not very dramatic, it should also be noted that the probability of finding the channel in an open state is larger at positive potentials than at negative potentials, following reconstitution of the isolated 106-kDa protein (Fig. 8A vs 8B; 8E vs 8F). In other reconstitution experiments, carried out with the isolated 106-kDa protein, a voltage asymmetry was always evident. However, depending on the orientation of the protein upon incorporation, in some experiments the channel activity was larger at more negative potentials. This asymmetric voltage dependence was present either before or after activation by rose bengal. It has also previously been reported following fusion of SR vesicles to a BLM (20). The single channel conductance in the presence of symmetric solutions of 250 mM NaCl, following fusion of SR vesicles to a bilayer (y = 400 pS; not shown) is the same as observed following incorporation of the isolated 106-kDa protein into the BLM (Figs. 7A, 8A).
ET AL.
H,O is between 2 and 5 pus,and is considerably longer in organic solvents (21). Given the high diffusion coefficient of O2 in water (D = lo-’ m2 s-l), it is likely that ‘02 diffuses at least 10 times the thickness of the bilayer before decaying. It is therefore not surprising that photoactivation of the release channel is independent of the side to which the rose bengal is added (Fig. 4). The Ca2+ release protein from skeletal muscle sarcoplasmic reticulum has previously been shown to be highly cation selective (13). However, the channel only slightly distinguishes between monovalent and divalent cations (15). The use of a high concentration CsCl gradient simplifies our analysis. Under these conditions, the SR K’ channel was strongly inhibited (16). At positive membrane potentials, near the reversal potential for chloride, Clfluxes were relatively insignificant, and we were then able to focus on Cs+ transport through the Ca2+ release channel. The Cs+ conducting Ca2+ release channel was inhibited by ruthenium red, and was activated by rose bengal
F
90 ms
DISCUSSION
Exposure of the photooxidizing dye rose bengal to light results in the production of ‘02, and the rapid release of Ca2+ from skeletal muscle SR vesicles (19). In this paper we show that the Ca2+ release channel from SR is activated by rose bengal in the presence of light (P,, increases), but the single channel conductance is unaffected. The nature of the activation is independent of the side to which rose bengal is added. The lifetime of singlet oxygen in
1 IOPA FIG. 8. Ryanodine-induced subconductance state is reversed by rose bengal. The 106-kDa protein was reconstituted into a BLM as described in the legend to Fig. 7. (A) Control trace at +40 mV, P, = 0.99. (B) Control trace at -40 mV, P, = 0.92. (C) Following addition of 10 nM ryanodine to both chambers at +40 mV, and (D) at -40 mV. (E) Addition of 500 nM rose bengal to both sides of the BLM in the presence of light. Recorded at +40 mV. P, = 0.93. (F) Same as (E) at -40 mV. P, = 0.12 (72 = 3).
Ca2+ RELEASE
FROM
SKELETAL
MUSCLE
in the light (Fig. 1). The rose bengal-activated channel did not show spontaneous inactivation. Following light exposure for 15 to 30 s, the channel was stable in the dark for periods of greater than 30 min. Neither the cation selectivity of the Ca2’ release channel (Fig. 1) nor the selectivity of the background current was modified by rose bengal. This latter observation indicated that no additional transport systems with different cation versus anion selectivity (i.e., K+ or Cl- channels) were being activated. This was confirmed in Fig. 2, in which K+ channels were shown to be unaffected by rose bengal and light. Activation of the relea.se channel from SR by adenine nucleotides (22,23), ryanodine (18), and doxorubicin (2426) has been shown to be Ca2+ dependent. Maximal activation occurs between 1 and 10 pM Ca2+. At Ca2+ concentrations as low as 10 nM the channel has been shown to be totally inhibited (22). Rose bengal-induced Ca2+ release and rose bengal activation of the Ca2’ release channel are both observed to be insensitive to Ca” (Figs. 2, 3). It is likely that the molecular mechanism underlying rose bengal-induced Ca” release is different than previously observed with other activating reagents. As shown in the previous paper (1!3), rose bengal-induced Cazf release is insensitive to Mg’+, while other methods of stimulating Ca2+ release are inhibited by millimolar concentrations of Mg2+ (22-24, 27-29). Furthermore, ATP has been shown to stimulate Ca2+ release from SR vesicles and to stimulate the Cazt release channel (22, 23). In the previous study, AMP-PCP, a nonhydrolyzable analog of ATP was shown to inhibit rose bengal-induced Ca2+ release from SR vesicles (19). In spite of this apparent difference in the mechanism of interaction with the -release channel, it is clear from this and the previous study that rose bengal interacts directly with the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum. Ryanodine’s ability to lock the release channel in a subconductance state (Fig. 5) was reversed upon addition of rose bengal, as was high affinity bound [3H]ryanodine (Fig. 6). Also, both the fusion studies with SR vesicles and the reconstitution experiments with the isolated 106-kDa sulfhydryl-activated protein indicate that rose bengal directly activated the Ca2+ release channel protein from SR. In comparing the properties of SR vesicles following fusion to a BLM and those of the reconstituted isolated 106-kDa protein most characteristics are similar. In both types of experiments, we observed a stimulation of channel activity upon addition of rose bengal and introduction of light (Figs. 1,7). In both cases, rose bengal was observed to reverse ryanodine modification of the Ca2+ release channel (Figs. 5, 8). Also, the conductance of the release channel in both the isolated 106-kDa protein preparation and the Ca2+ release channel following fusion of a SR vesicle to a BLM was identical. The differences between the two types of reconstitution experiments were more subtle. The sensitivity of the 106kDa protein to both rose bengal and ryanodine was greater
SARCOPLASMIC
RETICULUM
527
than that of the BLM following fusion of the SR. Where 10 nM ryanodine locked the 106-kDa protein in a subconductance state (Fig. 8), higher concentrations were needed to produce a similar effect following fusion to a BLM (Fig. 5). A concentration of 10 nM ryanodine slowed the gating of the release channel following fusion, but did not modify its conductance following fusion (30, 31). Concentrations of ryanodine slightly less than 1 pM also caused the channel following fusion to show subconductante states (Fig. 5). In a previous study, we observed that a minimum of 100 nM ryanodine was required in order to observe a subconductance state following fusion of SR vesicles to a BLM (31). The only experiments in which we noted a stimulation of channel activity by 500 nM rose bengal in the dark were carried out with the reconstituted 106-kDa protein (Fig. 7). In this experiment, we also observed a further activation by the addition of light. At the same rose bengal concentration, a stimulation of channel activity was not observed in the dark in the experiments carried out following fusion of SR vesicles to a BLM. In all SR fusion experiments in which rose bengal (500 nm1 pM) was added to either side of the BLM, activation of channel activity was observed within 20-30 s following exposure to light. Aside from the greater sensitivity of the reconstituted 106-kDa protein to ryanodine and rose bengal, the properties of the isolated protein and the SR following fusion to the BLM were strikingly similar. There was a strong correlation between the properties of the isolated 106kDa protein and the characteristics of rose bengal-stimulated Ca2+ release from SR vesicles. The site of action of photoinduced modification to the SR appears to be the 106-kDa sulfhydryl-activated Ca2+ channel protein. The fact that the characteristics of the 106-kDa protein and the SR following fusion agree so well gives strong credence to the identification of the 106-kDa protein as the Ca2+ release protein from sarcoplasmic reticulum. The sarcoplasmic reticulum has previously been implicated as a site of action for reactive oxygen damage. However, previous studies have focused on the Ca’+, Mg2+-ATPase as the primary target. This and the previous paper demonstrate that the Ca2+ release pathway is more sensitive to ROS damage than is the Ca2+ pump. Moreover, rapid Ca2+ release is induced by nanomolar concentrations of rose bengal in the presence of light by directly interacting with the ryanodine receptor, Ca2+ release protein, 106-kDa sulfhydryl-activated channel protein. A recent abstract by Cumming et ~2. (32) indicates that sheep cardiac SR shows a sensitivity to rose bengal similar to that of skeletal muscle SR. Whether or not the cardiac SR Ca2+ release protein is the primary site of action for ROS damage during reperfusion of cardiac muscle will require further detailed studies. REFERENCES 1. Manning, 497-518.
A. S., and Hearse, D. J. (1984) J. Mol. Cell. Cardiol.
16,
528
XIONG
2. Hess, M. L., and Manson, N. H. (1984) J. Mol. Cell. Cardiol. 16, 969-985. 3. Kukreja, R. C., Okabe, E., Schrier, G. M., and Hess, M. L. (1988) Arch. Biochem. Biophys. 261,441-457. 4. Kim, M-S., and Akera, T. (1987) Am. J. Physiol. 252, H252-H257. 5. MacLennan, D. H. (1970) J. Biol. Chem. 245,4508-4518. 6. Salama, G., and Abramson, J. J. (1984) J. Biol. Chem. 259, 13,36313,369.
7. Inui, M., Saito, A., Fleischer, S. (1987) J. Biol. Chem. 262, 17401747.
8. Zaidi, N. F., Lagenaur, C. F., Hilkert, 9. 10. 11.
12. 13. 14. 15. 16. 17. 18.
R. J., Xiong, H., Abramson, J. J., and Salama, G. (1989) J. Biol. Chem. 264, 21,737-21,747. Pessah, I. N., Stambuk, R. A., and Casida, J. E. (1987) Mol. Phurmacol. 31,232-238. Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505. Miller, C., Bell, J. E., and Garcia, A. M. (1984) in Current Topics in Membranes and Transport (Stein, W. D., Ed., Vol. 21, pp. 99132, Academic Press, New York. Rousseau, E. (1989) J. Membr. Biol. 110, 39-47. Smith, J. S., Coronado, R., and Meissner, G. (1985) Nature 316, 446-449. Ohnishi, S. T. (1979) J. Biochem. 86, 1147-1150. Smith, J. S., Imagawa, T., Ma, J., Fill, M., Campbell, K. P., and Coronado, R. (1988) J. Gen. Physiol. 92, l-26. Coronado, R., and Miller, C. (1979) Nature 280,807-810. Smith, J. S., Coronado, R., and Meissner, G. (1986) Biophys. J. 50, 921-928. Rousseau, E., Smith, J. S., and Meissner, G. (1987) Am. J. Physiol.
253,C364-C368.
ET AL. 19. Stuart, J., Pessah, I. N., Favero, T. G., and Abramson, Arch. Biochem. Biophys. 292, 512-521.
J. J. (1991)
20. Ma, J., Fill, M., Knudson, C. M., Campbell, K. P., and Coronado, R. (1988) Science 242, 99-102.
21. Perez, H. D. (1985) in Handbook Research (Greenwald, Raton, FL.
of Methods for Oxygen Radical R. A., Ed.), pp. 111-113, CRC Press, Boca
22. Smith, J. S., Coronado, R., and Meissner, G. (1986) J. Gen. Physiol. 88,573-588. 23. Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374. 24. Abramson, J. J., Buck, E., Salama, G., Casida, J. E., and Pessah, I. N. (1988) J. Biol. Chem. 263, l&750-18,758.
25. Holmberg, S. R. M., and Williams, A. J. (1990) Circ. Res. 67, 272283. 26. Ondrias, K., Borgatta, L., Kim, D. H., and Ehrlich, B. E. (1990) Circ. Res. 67, 1167-1174.
27. Salama, G., and Abramson, J. (1984) J. Biol. Chem. 259, 13,36313,369.
28. Trimm, J. L., Salama, G., and Abramson, J. J. (1986) J. Biol. Chem. 261, 16,092-16,098. 29. Abramson, J. J., Cronin, J., and Salama, G. (1988) Arch. Biochem. Biophys. 263, 245-255.
30. Bull, R., Marengo,
J. J., Suarez-Isla, B. A., Donoso, J. L., and Hidalgo, C. (1989) Biophys. J. 56, 749-756.
31. Zimanyi, Submitted
I., Buck, E., Abramson, for publication.
32. Cumming, D., Holmberg,
P., Sutko,
J. J., and Pessah, I. N. (1991)
S., Kusama, Y., Shattock, liams, A. (1990) J. Physiol. 420, 88P.
M., and Wil-
