ARCHIVRS
OF HIDCHEMISTRY
AND
BIOPHYSICS
Vol. 278, No. 1, April, pp. 251-257, 1990
Stimulation and Inhibition of [3H]Ryanodine Binding to Sarcoplasmic Reticulum from Malignant Hyperthermia Susceptible Pigs’ James R. Mickelson,’
Lynn
A. Litterer,
Department of Veterinary Biology, University St.-Paul, Minnesota 55108
Received Septemher
131989,
Blake
A. Jacobson,
of Minnesota,
and in revised form November
295 Animal
Press,
Inc.
’ This work was supported GM-31382. ’ To whom correspondence 0003-9861/90
by National
Institutes
should he addressed.
$3.00
Copyright (c 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Health
F. Louis Medicine,
28,1989
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of PM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (& = 6.0 nM) or 10 mM caffeine (& = 28 nM) was significantly greater than that of the normal SR (& = 8.5 and 65 nM in 10 mM ATP or caffeine, respec(12-16 pmol/mg) was similar in all tively); the B,,, cases. The CG% for inhibition of 13H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 pM for MHS and normal SR, respectively) and the Cd: for stimulation of t3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 pM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C& of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (CD.5 of 1.9 vs 1.2 FM) or Mg2+ inhibition (C,,, of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR. G,isso Academic
and Charles Science/Veterinary
Grant
A significant abnormality exists in the regulation of sarcoplasmic reticulum (SR)” calcium release in muscle of individuals susceptible to the pharmacogenetic disorder malignant hyperthermia (MH). This defect has been observed as both an increased sensitivity to contractures induced by caffeine and/or halothane in muscle biopsies (1,2) and an increased rate of calcium release in skinned single muscle fibers (3, 4) from MH susceptible (MHS) individuals (humans and pigs). Furthermore, isolated SR preparations from MHS pig muscle have been shown to exhibit both a lower intravesicular calcium threshold for the initiation of calcium efflux (5, 6) and a greater rate constant for calcium efflux when stimulated by Ca2+ and a variety of other agents (7, S), than does SR from normal pig muscle. The close association between MH and the alteration in SR calcium release is further demonstrated by the observation that SR from pigs heterozygous for the gene responsible for MH demonstrates a rate of calcium release intermediate between the SR of MHS (homozygous recessive) and normal (homozygous normal) pigs (9). An abnormality in the regulation of SR calcium release appears to be the primary defect in MHS muscle, accounting for the elevated sarcoplasmic Ca2+ concentration (10, ll), the muscle contracture (12, 13), and the significant stimulation of metabolism (12, 13) that typifies the MH response. The SR calcium release channel, which binds the plant alkaloid ryanodine with high affinity (14-16), is composed of junctional feet structures which bridge the gap between the SR terminal cisternae and the transverse tubules (17-19). When compared to normal SR, ’ Abbreviations used: MH, malignant hyperthermia; MHS, malignant hyperthermia susceptible; SR, sarcoplasmic reticulum; PMSF, phenylmethylsulfonyl fluoride; EGTA, [ethylenehis(oxyethylenenitrilolltetraacetic acid, Pipes, 1,4-piperazinediethanesulfonic acid, AMPPNP, adenylyl imidodiphosphate; C,,,, concentration for halfmaximal effect. 251
252
MICKELSON
the calcium channellryanodine receptor of MHS porcine SR demonstrates a significantly higher apparent affinity for [“Hlryanodine in the presence of PM Cap+ (8). In addition, MHS SR displays an altered Ca2’ dependence of [3H]ryanodine binding, particularly at the lowaffinity Ca2+ site which inhibits [“Hlryanodine binding (8). Ryanodine appears to bind to its receptor when the calcium channel is open (20-22). As shown by studies measuring calcium release from SR vesicles, or single release channel activity in planar lipid bilayers, this state is promoted by PM Ca2’ and inhibited by higher (mM) Ca” (23-28). Thus, an increased open state probability of the MHS SR calcium release channel, due to a reduced Ca2’ -dependent inactivation, could explain both the increased rate of calcium efflux and the increased affinity for ryanodine of MHS SR. Such a proposal is supported by our recent study of the single channel kinetics of the MHS SR calcium channel (26). It is therefore apparent from a number of studies that the Ca”+-dependent regulation of the SR calcium release channel is altered in MH. However, it is not yet known if the interaction of other compounds which can regulate this channel, such as adenine nucleotides, caffeine, ruthenium red, and Mg2+, is also altered in MHS SR. In this study, we have utilized [“H Jryanodine binding to its receptor as a reporter of the calcium release channel open state, and have noted a number of differences between MHS and normal SR [“Hlryanodine binding in their response to these regulatory agents. These results not only clarify the nature of the MHS SR calcium channellryanodine receptor defect, but. also provide insight into the mechanism of the normal SR calcium release process. EXPERIMENTAL
PROCEDURES
Materials. Pigs (29-45 kg) were obtained from the University of Minnesota Experimental Farm, where they were part of a swine genetics herd maintained by Dr. Bill Rempel for studies of the inheritance of halothane sensitivity. All Pietrain (homozygous for the halothanesensitivity gene) and Yorkshire (homozygous for the normal allele) pigs were halothane tested for MH susceptibility (29). The pigs were brought to the laboratory no sooner than 2 weeks after the halothanechallenge test. Unlabeled ryanodine was obtained from Calbiochem, and [“HIryanodine was purchased from DuPont New England Nuclear. Heavy SR membranes were preSarcoplasmic reticulum isolation. pared from the longissimus dorsi muscle as described previously (8, 9, 30). Briefly, the membrane pellet obtained by centrifugation of the muscle homogenate between 2600 and 10,OOOgwas resuspended in 0.6 M KC1 buffer, incubated on ice for 1 h, and centrifuged at 100,OOOg. The pellets were resuspended and placed on discontinuous sucrose density gradients. The membranes which banded at the 36-45s sucrose interface after 5 h centrifugation at 85,OOOgwere collected and used in all experiments. All solutions contained 0.1 mM PMSF, 1 rg/ ml pepstatin, 1 *g/ml leupeptin, 1 pg/ml aprotinin, and 0.8 mM benzamidine to minimize proteolysis. binding was per[3H]Ryanodine binding assay. [“H]Ryanodine formed for 90 min at 37°C in media containing 0.2 mg SR protein/ml, 0.1 M KCI, 10 mM Pipes (pH 7.0), 0.5 mM PMSF, 10 nM [“Hlryanodine,
ET AL. 16
8
E 2
14
g s
12
0
,
Bound I
I
0
100
I
200
I
300
do
560
&O
[Ryanodine] (nM) FIG. 1. [“H]Ryanodine binding to MHS (w) and normal (0) SR in binding the presence of 1 pM Ca’+ and 10 mM caffeine. [JH]Ryanodine was performed as described under Experimental Procedures. Inset; Scatchard plots. Symbols represent means 4 SE of values determined for three different MHS or four different normal SR preparations.
and a CaCl,-EGTA-nitrilotriacetic acid buffer set to give various defined Ca2+ concentrations (8, 9). The final ryanodine concentration (up to 600 nM) was achieved by addition of unlabeled ryanodine; other compounds were present as indicated. We have previously demonstrated that for the porcine SR, 90 min incubation was sufficient for [“Hlryanodine binding to reach equilibrium (9). Samples were filtered onto Whatman GF/B filters using a Brandel M-24R cell harvester, and the filters were washed with 10 ml ice-cold 0.1 M KCl, 10 mM Pipes (pH 7.0) buffer. Specific [“Hlryanodine binding was determined by subtracting the nonspecific binding obtained in the presence of 20 pM unlabeled ryanodine. From the [“Hlryanodine binding data, Scatchard plots were constructed to obtain both the ryanodine affinity (&), and content (R,,,,) of the ryanodine receptor. Hill plots were utilized to determine the concentration of added agent which resulted in half-maximal stimulation or inhibition (C,,) of [“Hlryanodine binding. All lines were fit by linear regression analysis. Only the binding data from which the Hill plots were prepared is shown in the figures. Statistical analyses were performed with Student’s t test, utilizing SR preparations from at least three different MHS and normal pigs.
RESULTS
of the Ryanodine Receptor in the Presence of Ca”+, Plus Either Caffeine or A TP
Kd and B,,,
We have previously demonstrated that at optimal Cazi (6 PM) MHS SR displays a significantly higher apparent affinity for ryanodine than does normal SR; however, there was no difference in the B,,, of the MHS and normal SR ryanodine receptor under these conditions (8) (See also Table I). Figures 1 and 2 show that in the presence of 10 mM caffeine (Ca’+ of 1 PM) or 10 mM ATP (Ca’+ of 6 PM) MHS and normal SR demonstrated saturable [“Hlryanodine binding; the Kd and B,,, values are reported in Table I. Both caffeine and ATP lowered the apparent Kd of the ryanodine receptor for ryanodine,
SARCOPLASMIC
RETICULUM
RYANODINE
253
RECEPTOR
N MHS
-0 I 2
1
2 z 0 0
30
10
40
10-2
[Ryanociin:~ (nM)
with ATP exerting the larger effect. In the presence of caffeine, the Kd of the MHS SR for ryanodine was significantly lower than that of the normal SR, while the difference in the Kd values between MHS and normal SR in the presence of ATP was small, although statistically significant (Table I). The B,,, values obtained for the MHS and normal SR ryanodine receptor were similar under these different conditions. Binding
in the
In our previous study we demonstrated that the Ca2+ dependence for both the stimulation and the inhibition
TABLE
I
Kd and B,,, Values of the [“H]Ryanodine
Receptor of MHS and Normal SR Obtained Under Various Conditions Kd MHS
6lMCa”
‘I
1 PM
t
10 6&M
10
Ca” mM
Ca” mM
Caffeine’ t ATPd
92
‘-7h
(nM)
ha,
Normal 265
100
IO'
102
[Calcium] (PM)
FIG. 2. [“HJRyanodine binding to MHS (m) and normal (0) SR in binding the presence of 6 FM Ca” and 10 mM ATP. c’H]Ryanodine was performed as described under Experimental Procedures. Inset; Scatchard plots. Symbols represent the means of values determined for five different MHS or normal SR preparations.
Ca” Dependence of [“H]Ryanodine Presence of Caffeine or AMPPNP
10-l
bmol/md
MHS
t 28
Normal
12.6 11.1
9.3 i 1.3
28.5 -+ 3.Th
65.0?
5.3
14.010.6
12.1 + 1.2
6.0 f 0.4h
8.5 i
0.3
14.8 t 1.5
15.9? 0.9
I’ As reported by Mickelson, et al. (8). ’ Significantly different from normal values P < 0.01. ’ Means 2 SE, calculated from the data obtained for each individual preparation utilized in Fig. 1. ’ Means + SE, calculated from the data obtained for each individual preparation utilized in Fig. 2.
FIG. 3. Ca” dependence of [“Hlryanodine binding to MHS (m) and normal (0) SR in the presence of 5 mM caffeine. [:‘H]Ryanodine binding was performed at a ryanodine concentration of 10 nhq as described under Experimental Procedures. Symbols represent means + SE of values determined for four different MHS or three different normal SR preparations.
of [“Hlryanodine binding to MHS SR was altered, with slightly lower Ca”+ required to stimulate and substan[“Hlryanodine tially higher Ca2+ required to inhibit binding to MHS, when compared to normal SR (8) (see also Table II). The effect of caffeine and AMPPNP on the Ca”’ dependence of MHS and normal SR [“HIryanodine binding is shown in Fig. 3 and 4 respectively, for a ryanodine concentration of 10 nM. The Ca”’ dependencies of [3H]ryanodine binding in both cases could be described by a bell-shaped curve, with optimal Ca” of 0.1-1.0 PM in the presence of 5 mM caffeine, and 6-10 PM in the presence of 5 mM AMPPNP. As observed previously in the absence of these agents (8), [“Hlryanodine binding to MHS SR was greater than that to normal SR at any given Ca2+ concentration. When the Caif values for the stimulation of SR [“HIryanodine binding are compared to those obtained in the absence of any added agent, it is seen that caffeine substantially lowered the Ca,“,:? for stimulation of [“HIryanodine binding to both MHS and normal SR, while AMPPNP had little effect on this value for either type of SR (Table II). In the presence of either caffeine or AMPPNP, the CaiA for stimulation of [“Hlryanodine binding to MHS SR was significantly lower than that for the stimulation of t3H]ryanodine binding to normal SR (Table II). The Hill coefficient (n) for Ca2’ stimulation was 1.3-1.6 in the presence of caffeine or AMPPNP, with no difference between MHS and normal values. When the Cai: values for the inhibition of [“HIryanodine binding in the presence of caffeine or AMPPNP are compared to those in the absence of added agents, it is seen that AMPPNP significantly increased
254
MICKELSON
mal SR [“Hlryanodine binding in the presence of optimal Ca2+ concentrations (l-6 PM) are shown in Figs. 5was 7. As expected, binding at 100 nM [3H]ryanodine greater than that at 10 nM (compare Fig. 7 with Figs. 5 and 6A), while [3H]ryanodine binding was greater in the presence than in the absence of 10 mM ATP (compare Figs. 6B and 6A). Again, under any given set of conditions, MHS SR bound more [3H]ryanodine than did normal SR. Figure 5 demonstrates that in the presence of 1 pM Ca’+, caffeine stimulated both MHS and normal SR [3H]ryanodine binding by no more than twofold. However, less caffeine was required to stimulate [3H]ryanodine binding to MHS than to normal SR (Table III). In contrast, ruthenium red (Fig. 6) and Mg2+ (Fig. 7) inhibited [3H]ryanodine binding to both MHS and normal SR. Furthermore, more ruthenium red was required to inhibit [“Hlryanodine binding in the presence than in the absence of ATP. In the presence of ATP more ruthenium red was required to inhibit MHS than normal SR [3H]ryanodine binding, while in the absence of ATP MHS and normal SR [3H]ryanodine binding did not differ significantly in their response to ruthenium red. Table III also shows that more Mg2+ was required to inhibit [3H]ryanodine binding to MHS than was required to inhibit binding to normal SR. The Hill coefficient for caffeine stimulation of [3H]ryanodine binding was 0.8-1.0, while the Hill coefficient for inhibition of [3H]ryanodine binding varied from 1.6-2.0 for ruthenium red and from 0.8-1.0 for Mg*+; the Hill coefficients for MHS and normal SR did not differ.
6
[Calcium] (PM) FIG. 4. Ca’+ dependence of [“Hlryanodine binding normal (0) SR in the presence of 5 mM AMPPNP. binding was performed at a ryanodine concentration scribed under Experimental Procedures. Symbols k SE of values determined for four different MHS or arations.
to MHS (B) and [“H]Ryanodine of 10 nM, as derepresent means normal SR prep-
the Cai; for inhibition of [3H]ryanodine binding to both MHS and normal SR (Table II). Interestingly, caffeine either lowered or had no effect on this Cai:’ value for MHS and normal SR, respectively. In the presence of AMPPNP the Cd: for inhibition of [3H]ryanodine binding to MHS SR was greater than that for normal SR, while in the presence of caffeine MHS and normal SR CGi for inhibition of [3H]ryanodine binding were not significantly different. The Hill coefficient for Ca2+ inhibition was 0.8-1.0 in the presence of caffeine or AMPPNP, with no difference between MHS and normal values.
DISCUSSION This study compared the effects of Ca2+, adenine nucleotides, caffeine, ruthenium red, and Mg*+ on MHS and normal porcine SR [3H]ryanodine binding. The results not only confirm that an abnormality exists in the regulation of the calcium release channel/ryanodine receptor of MHS SR, but also provide insight into the regulation of the normal SR calcium release channel.
Concentration Dependent Effects of Caffeine, Ruthenium Red, and Mg2+ on [3H] Ryanodine Binding The effects of caffeine, ruthenium red (in the presence or absence of 10 mM ATP), and Mg2+ on MHS and norTABLE Ca*+
II
Dependence of [3H]Ryanodine Binding to MHS and Normal SR Stimulatory
Inhibitory
Ca”’ Normal
MHS cd: Ca’+ only” Ca2+ plus 5 mM caffeine’ Ca*+ plus 5 mM AMPPNPd
ET AL.
0.34 * 0.05 0.049 +- 0.005b 0.27 f 0.02'
a As reported by Mickelson, et al. (8). b Significantly different from normal values, P < 0.05. ’ Means ? SE, calculated from the data obtained for each individual d Means + SE, calculated from the data obtained for each individual
Normal
MHS c&
(Md
0.54 * 0.12 0.070 It 0.010 0.41 kO.06
preparation preparation
utilized in Fig. 3. utilized in Fig. 4.
Ca2+
52 t 10b 22* 2 238-t36*
(GM) 1814
15 *4 74t8
SARCOPLASMIC 5 ,
RETICULUM I
q
N
n
MHS
binding to MHS (m) and FIG. 5. Effect ofcaffeine on [“Hlryanodine normal (0) SR. [“Hlryanodine binding was performed at a Ca” concentration of 1 PM, and a ryanodine concentration of 10 nM, as described under Experimental Procedures. Symbols represent means i SE of values determined for three different MHS or normal SR preparations.
As previously noted by Pessah et al. (31), the apparent Kd of the ryanodine receptor for ryanodine depended upon the components present in the binding media (Figs. 1 and 2, Table I). The Kd observed in 6 PM Ca”+ was greater than that observed in the presence of yM Ca2’ plus 10 mM caffeine, which in turn was greater than that in the presence of 6 PM Ca”+ plus 10 mM ATP. This order of increasing ability of these agents to increase the apparent affinity for ryanodine correlates with their ability to increase the SR calcium channel open state
2,
RYANODINE
255
RECEPTOR
probability (23-27, 32); i.e., the experimentally measured affinity of the receptor for ryanodine is dependent upon the open state probability of the channel. Although the kinetics of [3H]ryanodine binding to its receptor (31, 33) and its effect on calcium channel activity are complex (14-16,20-22,25), these results are consistent with those of previous studies suggesting that ryanodine associates with its receptor when the channel is in the open state (20-22, 31). In all three conditions examined, the apparent Kd of the MHS SR receptor for ryanodine was significantly less than that for normal SR, although in the presence of Ca’?+ plus ATP the magnitude of the difference was quite small (Table I). Thus, when the calcium release channel is maximally activated (P,, of approximately 1 (24)), the apparent affinities of the MHS and normal SR for [“Hlryanodine are essentially identical; only when the channel is partially active (i.e., PO of less than 1) are differences in the apparent affinity of the MHS and normal ryanodine receptor for [3H]ryanodine readily detectable. The L,, of the porcine ryanodine receptor did not vary significantly under the different incubation conditions and was similar for both MHS and normal SR (Table I). This confirms our earlier observation that the calcium release channel protein content is not significantly different in MHS and normal SR (8,9). Thus, a greater MHS SR channel content cannot explain the reports of an increased rate of MHS SR calcium efflux (3, 4, 79). Rather, differences in the regulation of the MHS SR channel open state, allowing a greater P, or calcium conductance, are more likely responsible for increased rates of MHS SR calcium release. The Ca’+ dependence of [“Hlryanodine binding to both MHS and normal SR in the presence of 5 mM
I q
N
n
MHS
6
[Ruthenium Red] (M)
[Ruthenium Red] (M)
binding to MHS (D) and normal (0) SR. [“Hlryanodine binding was performed at a Ca” FIG. 6. Effect of ruthenium red on [“Hlryanodine concentration of 6 pM, and a ryanodine concentration of 10 nM, in the absence (A) or presence (B) of 10 mM ATP. Symbols represent means t SE of values determined for four different MHS or normal SR preparations.
256
MICKELSON
4
2
0 2
[Magnesium] (mM) FIG. 7. EfIect of Mg*+ on [“Hlryanodine binding to MHS (a) and normal (0) SR. [“H]Ryanodine binding was performed at a Ca2+ concentration of 6 pM and a ryanodine concentration of 100 nM, as described under Experimental Procedures. Symbols represent means f SE of values determined for four different MHS or normal SR preparations.
caffeine or 5 mM AMPPNP exhibited a maxima in the PM Ca’+ range (Figs. 3 and 4). At any given Ca2+ concentration [“Hlryanodine binding to MHS SR was greater than to normal SR, likely due to the lower apparent Kd of the MHS SR receptor for ryanodine (Table I). The Caii for the stimulation and inhibition of SR [“HJryanodine binding varied depending on the components present in the binding media (Table II, see also (31,34)). Whereas AMPPNP did not alter the Cai,: for stimulation of [“Hlryanodine binding, caffeine significantly lowered this value. In contrast, the Caii for the inhibition of normal SR [“Hlryanodine binding was significantly increased by AMPPNP, but unaffected by caffeine. Thus, the effect of adenine nucleotide in raising the Cai,; for inhibition of [“Hlryanodine binding may be explained by AMPPNP inhibiting the channel Ca’-dependent inactivation mechanism since ryanodine appears to bind to the open state of the channel. In contrast, the lowering of the Cai: for stimulation of [3H]ryanodine binding by caffeine may be explained by caffeine stimulating the channel Ca” dependent activation mechanism. While there was an approximately 1.5-fold difference between MHS and normal SR in the Ca$: for activation of binding in the presence of Ca’+, Ca2+ plus AMPPNP, or Ca2+ plus caffeine (Table II), this was less significant than the up to 3-fold difference in the Cai; for the inhibition of binding in the presence of Ca2+, or Ca”’ plus AMPPNP. The major difference between MHS and normal SR [“Hlryanodine binding at high Ca”+ may be related to the abnormal kinetics of channel inactivation we have observed when this channel is inserted in planar lipid
ET AL
bilayers (26). In these experiments MHS channels failed to inactivate in millimolar Ca2+ concentrations (either cis or tram), resulting in a greater POfor the MHS channel (26). In the presence of PM Ca2+, caffeine stimulated, while ruthenium red and Mg” inhibited, [3H]ryanodine binding to the porcine SR (Figs. 5-7, Table III); this correlates with the ability of these agents to stimulate and inhibit respectively, both SR calcium release and single channel activity (23-25, 28, 32, 35). Interestingly, the sensitivity of SR [“Hlryanodine binding to ruthenium red inhibition was decreased in the presence of the stimulatory agent ATP (Table III). When the effects of these agents on MHS and normal SR [3H]ryanodine binding were compared it was found that less caffeine was required to stimulate, and more ruthenium red or Mg2+ was required to inhibit [3H]ryanodine binding to MHS SR than to normal SR. The small but significant difference in C0,6 for caffeine stimulation of [“Hlryanodine binding between MHS and normal SR correlates with the small difference we have observed in the C,, for caffeine stimulation of calcium release from MHS and normal SR vesicles (9). In all cases, however, there was at most a twofold difference in the C,, values for effects of caffeine, ruthenium red, and Mg2+ on [3H]ryanodine binding; the Cai: values for inhibition of [“Hlryanodine binding (Table II) and the Kd for ryanodine in 6 PM Ca”+ (Table I) demonstrated the largest difference in any value between MHS and normal SR. We conclude that the altered Ca2+ regulation of the calcium release channel (3-9,26,30) may be the primary
TABLE Sensitivity of MHS to Stimulation
III
and Normal SR t3H]Ryanodine Binding or Inhibition by Various Agents Concentration for half-maximal effect (C,,,) MHS
Agent Caffeine (mM) (+l pM Ca”‘)” Ruthenium red (PM) (+6 gM Ca”‘)’ Ruthenium red (PM) (+lO mM ATP) (+6 pM Cay’)’ Mg” (mM) (t6 fiM CZL”)~
1.7
t0.3*
Normal 3.4
* 0.2
0.079 f 0.009
0.083 f 0.011
1.9 kO.2” 0.34 * o.ofJ*
1.2 + 0.1 0.21 + 0.01
UMeans 2 SE for stimulation of [“Hlryanodine binding, calculated from the data obtained for each individual preparation utilized in Figure 5. ’ Significantly ditferent from normal values, P < 0.05. ’ Means 2 SE for inhibition of 13H]ryanodine binding, calculated from the data obtained for each individual preparation utilized in Fig. 6. “Means k SE for inhibition of [“Hlryanodine binding, calculated from the data obtained for each individual preparation utilized in Fig. 7.
SARCOPLASMIC
RETICULUM
abnormality in MHS SR, which may then result in subsequent alterations in the interaction of other ligands such as caffeine and halothane with the MHS channel, both in isolated SR (5-9,30,36) and in more intact muscle preparations (l-4). A possible explanation for our results is that there is a structural alteration in the Ca” binding domain(s) that control(s) the gating properties of the MHS calcium channel protein. However, modifications in channel accessory/regulatory proteins, including a decreased extent of phosphorylation of a 60kDa protein in MHS SR believed to play a role in regulating calcium release (37), are still possible. Definition of the molecular basis of the alterations in the MHS muscle ryanodine receptor, as well as the dihydropyridine receptor (38), should provide insight into both the mechanism of excitation-contraction coupling, and the diagnosis and treatment of this significant muscle disorder. ACKNOWLEDGMENTS \Ve thank Drs. Esther Gallant and ,Jim Ervasti for helpful discussion and Dr. William Rempel for the supply of experimental animals.
RYANODINE
257
RECEPTOR
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OF HIDCHEMISTRY
AND
BIOPHYSICS
Vol. 278, No. 1, April, pp. 251-257, 1990
Stimulation and Inhibition of [3H]Ryanodine Binding to Sarcoplasmic Reticulum from Malignant Hyperthermia Susceptible Pigs’ James R. Mickelson,’
Lynn
A. Litterer,
Department of Veterinary Biology, University St.-Paul, Minnesota 55108
Received Septemher
131989,
Blake
A. Jacobson,
of Minnesota,
and in revised form November
295 Animal
Press,
Inc.
’ This work was supported GM-31382. ’ To whom correspondence 0003-9861/90
by National
Institutes
should he addressed.
$3.00
Copyright (c 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
of Health
F. Louis Medicine,
28,1989
When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of PM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (& = 6.0 nM) or 10 mM caffeine (& = 28 nM) was significantly greater than that of the normal SR (& = 8.5 and 65 nM in 10 mM ATP or caffeine, respec(12-16 pmol/mg) was similar in all tively); the B,,, cases. The CG% for inhibition of 13H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 pM for MHS and normal SR, respectively) and the Cd: for stimulation of t3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 pM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C& of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (CD.5 of 1.9 vs 1.2 FM) or Mg2+ inhibition (C,,, of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR. G,isso Academic
and Charles Science/Veterinary
Grant
A significant abnormality exists in the regulation of sarcoplasmic reticulum (SR)” calcium release in muscle of individuals susceptible to the pharmacogenetic disorder malignant hyperthermia (MH). This defect has been observed as both an increased sensitivity to contractures induced by caffeine and/or halothane in muscle biopsies (1,2) and an increased rate of calcium release in skinned single muscle fibers (3, 4) from MH susceptible (MHS) individuals (humans and pigs). Furthermore, isolated SR preparations from MHS pig muscle have been shown to exhibit both a lower intravesicular calcium threshold for the initiation of calcium efflux (5, 6) and a greater rate constant for calcium efflux when stimulated by Ca2+ and a variety of other agents (7, S), than does SR from normal pig muscle. The close association between MH and the alteration in SR calcium release is further demonstrated by the observation that SR from pigs heterozygous for the gene responsible for MH demonstrates a rate of calcium release intermediate between the SR of MHS (homozygous recessive) and normal (homozygous normal) pigs (9). An abnormality in the regulation of SR calcium release appears to be the primary defect in MHS muscle, accounting for the elevated sarcoplasmic Ca2+ concentration (10, ll), the muscle contracture (12, 13), and the significant stimulation of metabolism (12, 13) that typifies the MH response. The SR calcium release channel, which binds the plant alkaloid ryanodine with high affinity (14-16), is composed of junctional feet structures which bridge the gap between the SR terminal cisternae and the transverse tubules (17-19). When compared to normal SR, ’ Abbreviations used: MH, malignant hyperthermia; MHS, malignant hyperthermia susceptible; SR, sarcoplasmic reticulum; PMSF, phenylmethylsulfonyl fluoride; EGTA, [ethylenehis(oxyethylenenitrilolltetraacetic acid, Pipes, 1,4-piperazinediethanesulfonic acid, AMPPNP, adenylyl imidodiphosphate; C,,,, concentration for halfmaximal effect. 251
252
MICKELSON
the calcium channellryanodine receptor of MHS porcine SR demonstrates a significantly higher apparent affinity for [“Hlryanodine in the presence of PM Cap+ (8). In addition, MHS SR displays an altered Ca2’ dependence of [3H]ryanodine binding, particularly at the lowaffinity Ca2+ site which inhibits [“Hlryanodine binding (8). Ryanodine appears to bind to its receptor when the calcium channel is open (20-22). As shown by studies measuring calcium release from SR vesicles, or single release channel activity in planar lipid bilayers, this state is promoted by PM Ca2’ and inhibited by higher (mM) Ca” (23-28). Thus, an increased open state probability of the MHS SR calcium release channel, due to a reduced Ca2’ -dependent inactivation, could explain both the increased rate of calcium efflux and the increased affinity for ryanodine of MHS SR. Such a proposal is supported by our recent study of the single channel kinetics of the MHS SR calcium channel (26). It is therefore apparent from a number of studies that the Ca”+-dependent regulation of the SR calcium release channel is altered in MH. However, it is not yet known if the interaction of other compounds which can regulate this channel, such as adenine nucleotides, caffeine, ruthenium red, and Mg2+, is also altered in MHS SR. In this study, we have utilized [“H Jryanodine binding to its receptor as a reporter of the calcium release channel open state, and have noted a number of differences between MHS and normal SR [“Hlryanodine binding in their response to these regulatory agents. These results not only clarify the nature of the MHS SR calcium channellryanodine receptor defect, but. also provide insight into the mechanism of the normal SR calcium release process. EXPERIMENTAL
PROCEDURES
Materials. Pigs (29-45 kg) were obtained from the University of Minnesota Experimental Farm, where they were part of a swine genetics herd maintained by Dr. Bill Rempel for studies of the inheritance of halothane sensitivity. All Pietrain (homozygous for the halothanesensitivity gene) and Yorkshire (homozygous for the normal allele) pigs were halothane tested for MH susceptibility (29). The pigs were brought to the laboratory no sooner than 2 weeks after the halothanechallenge test. Unlabeled ryanodine was obtained from Calbiochem, and [“HIryanodine was purchased from DuPont New England Nuclear. Heavy SR membranes were preSarcoplasmic reticulum isolation. pared from the longissimus dorsi muscle as described previously (8, 9, 30). Briefly, the membrane pellet obtained by centrifugation of the muscle homogenate between 2600 and 10,OOOgwas resuspended in 0.6 M KC1 buffer, incubated on ice for 1 h, and centrifuged at 100,OOOg. The pellets were resuspended and placed on discontinuous sucrose density gradients. The membranes which banded at the 36-45s sucrose interface after 5 h centrifugation at 85,OOOgwere collected and used in all experiments. All solutions contained 0.1 mM PMSF, 1 rg/ ml pepstatin, 1 *g/ml leupeptin, 1 pg/ml aprotinin, and 0.8 mM benzamidine to minimize proteolysis. binding was per[3H]Ryanodine binding assay. [“H]Ryanodine formed for 90 min at 37°C in media containing 0.2 mg SR protein/ml, 0.1 M KCI, 10 mM Pipes (pH 7.0), 0.5 mM PMSF, 10 nM [“Hlryanodine,
ET AL. 16
8
E 2
14
g s
12
0
,
Bound I
I
0
100
I
200
I
300
do
560
&O
[Ryanodine] (nM) FIG. 1. [“H]Ryanodine binding to MHS (w) and normal (0) SR in binding the presence of 1 pM Ca’+ and 10 mM caffeine. [JH]Ryanodine was performed as described under Experimental Procedures. Inset; Scatchard plots. Symbols represent means 4 SE of values determined for three different MHS or four different normal SR preparations.
and a CaCl,-EGTA-nitrilotriacetic acid buffer set to give various defined Ca2+ concentrations (8, 9). The final ryanodine concentration (up to 600 nM) was achieved by addition of unlabeled ryanodine; other compounds were present as indicated. We have previously demonstrated that for the porcine SR, 90 min incubation was sufficient for [“Hlryanodine binding to reach equilibrium (9). Samples were filtered onto Whatman GF/B filters using a Brandel M-24R cell harvester, and the filters were washed with 10 ml ice-cold 0.1 M KCl, 10 mM Pipes (pH 7.0) buffer. Specific [“Hlryanodine binding was determined by subtracting the nonspecific binding obtained in the presence of 20 pM unlabeled ryanodine. From the [“Hlryanodine binding data, Scatchard plots were constructed to obtain both the ryanodine affinity (&), and content (R,,,,) of the ryanodine receptor. Hill plots were utilized to determine the concentration of added agent which resulted in half-maximal stimulation or inhibition (C,,) of [“Hlryanodine binding. All lines were fit by linear regression analysis. Only the binding data from which the Hill plots were prepared is shown in the figures. Statistical analyses were performed with Student’s t test, utilizing SR preparations from at least three different MHS and normal pigs.
RESULTS
of the Ryanodine Receptor in the Presence of Ca”+, Plus Either Caffeine or A TP
Kd and B,,,
We have previously demonstrated that at optimal Cazi (6 PM) MHS SR displays a significantly higher apparent affinity for ryanodine than does normal SR; however, there was no difference in the B,,, of the MHS and normal SR ryanodine receptor under these conditions (8) (See also Table I). Figures 1 and 2 show that in the presence of 10 mM caffeine (Ca’+ of 1 PM) or 10 mM ATP (Ca’+ of 6 PM) MHS and normal SR demonstrated saturable [“Hlryanodine binding; the Kd and B,,, values are reported in Table I. Both caffeine and ATP lowered the apparent Kd of the ryanodine receptor for ryanodine,
SARCOPLASMIC
RETICULUM
RYANODINE
253
RECEPTOR
N MHS
-0 I 2
1
2 z 0 0
30
10
40
10-2
[Ryanociin:~ (nM)
with ATP exerting the larger effect. In the presence of caffeine, the Kd of the MHS SR for ryanodine was significantly lower than that of the normal SR, while the difference in the Kd values between MHS and normal SR in the presence of ATP was small, although statistically significant (Table I). The B,,, values obtained for the MHS and normal SR ryanodine receptor were similar under these different conditions. Binding
in the
In our previous study we demonstrated that the Ca2+ dependence for both the stimulation and the inhibition
TABLE
I
Kd and B,,, Values of the [“H]Ryanodine
Receptor of MHS and Normal SR Obtained Under Various Conditions Kd MHS
6lMCa”
‘I
1 PM
t
10 6&M
10
Ca” mM
Ca” mM
Caffeine’ t ATPd
92
‘-7h
(nM)
ha,
Normal 265
100
IO'
102
[Calcium] (PM)
FIG. 2. [“HJRyanodine binding to MHS (m) and normal (0) SR in binding the presence of 6 FM Ca” and 10 mM ATP. c’H]Ryanodine was performed as described under Experimental Procedures. Inset; Scatchard plots. Symbols represent the means of values determined for five different MHS or normal SR preparations.
Ca” Dependence of [“H]Ryanodine Presence of Caffeine or AMPPNP
10-l
bmol/md
MHS
t 28
Normal
12.6 11.1
9.3 i 1.3
28.5 -+ 3.Th
65.0?
5.3
14.010.6
12.1 + 1.2
6.0 f 0.4h
8.5 i
0.3
14.8 t 1.5
15.9? 0.9
I’ As reported by Mickelson, et al. (8). ’ Significantly different from normal values P < 0.01. ’ Means 2 SE, calculated from the data obtained for each individual preparation utilized in Fig. 1. ’ Means + SE, calculated from the data obtained for each individual preparation utilized in Fig. 2.
FIG. 3. Ca” dependence of [“Hlryanodine binding to MHS (m) and normal (0) SR in the presence of 5 mM caffeine. [:‘H]Ryanodine binding was performed at a ryanodine concentration of 10 nhq as described under Experimental Procedures. Symbols represent means + SE of values determined for four different MHS or three different normal SR preparations.
of [“Hlryanodine binding to MHS SR was altered, with slightly lower Ca”+ required to stimulate and substan[“Hlryanodine tially higher Ca2+ required to inhibit binding to MHS, when compared to normal SR (8) (see also Table II). The effect of caffeine and AMPPNP on the Ca”’ dependence of MHS and normal SR [“HIryanodine binding is shown in Fig. 3 and 4 respectively, for a ryanodine concentration of 10 nM. The Ca”’ dependencies of [3H]ryanodine binding in both cases could be described by a bell-shaped curve, with optimal Ca” of 0.1-1.0 PM in the presence of 5 mM caffeine, and 6-10 PM in the presence of 5 mM AMPPNP. As observed previously in the absence of these agents (8), [“Hlryanodine binding to MHS SR was greater than that to normal SR at any given Ca2+ concentration. When the Caif values for the stimulation of SR [“HIryanodine binding are compared to those obtained in the absence of any added agent, it is seen that caffeine substantially lowered the Ca,“,:? for stimulation of [“HIryanodine binding to both MHS and normal SR, while AMPPNP had little effect on this value for either type of SR (Table II). In the presence of either caffeine or AMPPNP, the CaiA for stimulation of [“Hlryanodine binding to MHS SR was significantly lower than that for the stimulation of t3H]ryanodine binding to normal SR (Table II). The Hill coefficient (n) for Ca2’ stimulation was 1.3-1.6 in the presence of caffeine or AMPPNP, with no difference between MHS and normal values. When the Cai: values for the inhibition of [“HIryanodine binding in the presence of caffeine or AMPPNP are compared to those in the absence of added agents, it is seen that AMPPNP significantly increased
254
MICKELSON
mal SR [“Hlryanodine binding in the presence of optimal Ca2+ concentrations (l-6 PM) are shown in Figs. 5was 7. As expected, binding at 100 nM [3H]ryanodine greater than that at 10 nM (compare Fig. 7 with Figs. 5 and 6A), while [3H]ryanodine binding was greater in the presence than in the absence of 10 mM ATP (compare Figs. 6B and 6A). Again, under any given set of conditions, MHS SR bound more [3H]ryanodine than did normal SR. Figure 5 demonstrates that in the presence of 1 pM Ca’+, caffeine stimulated both MHS and normal SR [3H]ryanodine binding by no more than twofold. However, less caffeine was required to stimulate [3H]ryanodine binding to MHS than to normal SR (Table III). In contrast, ruthenium red (Fig. 6) and Mg2+ (Fig. 7) inhibited [3H]ryanodine binding to both MHS and normal SR. Furthermore, more ruthenium red was required to inhibit [“Hlryanodine binding in the presence than in the absence of ATP. In the presence of ATP more ruthenium red was required to inhibit MHS than normal SR [3H]ryanodine binding, while in the absence of ATP MHS and normal SR [3H]ryanodine binding did not differ significantly in their response to ruthenium red. Table III also shows that more Mg2+ was required to inhibit [3H]ryanodine binding to MHS than was required to inhibit binding to normal SR. The Hill coefficient for caffeine stimulation of [3H]ryanodine binding was 0.8-1.0, while the Hill coefficient for inhibition of [3H]ryanodine binding varied from 1.6-2.0 for ruthenium red and from 0.8-1.0 for Mg*+; the Hill coefficients for MHS and normal SR did not differ.
6
[Calcium] (PM) FIG. 4. Ca’+ dependence of [“Hlryanodine binding normal (0) SR in the presence of 5 mM AMPPNP. binding was performed at a ryanodine concentration scribed under Experimental Procedures. Symbols k SE of values determined for four different MHS or arations.
to MHS (B) and [“H]Ryanodine of 10 nM, as derepresent means normal SR prep-
the Cai; for inhibition of [3H]ryanodine binding to both MHS and normal SR (Table II). Interestingly, caffeine either lowered or had no effect on this Cai:’ value for MHS and normal SR, respectively. In the presence of AMPPNP the Cd: for inhibition of [3H]ryanodine binding to MHS SR was greater than that for normal SR, while in the presence of caffeine MHS and normal SR CGi for inhibition of [3H]ryanodine binding were not significantly different. The Hill coefficient for Ca2+ inhibition was 0.8-1.0 in the presence of caffeine or AMPPNP, with no difference between MHS and normal values.
DISCUSSION This study compared the effects of Ca2+, adenine nucleotides, caffeine, ruthenium red, and Mg*+ on MHS and normal porcine SR [3H]ryanodine binding. The results not only confirm that an abnormality exists in the regulation of the calcium release channel/ryanodine receptor of MHS SR, but also provide insight into the regulation of the normal SR calcium release channel.
Concentration Dependent Effects of Caffeine, Ruthenium Red, and Mg2+ on [3H] Ryanodine Binding The effects of caffeine, ruthenium red (in the presence or absence of 10 mM ATP), and Mg2+ on MHS and norTABLE Ca*+
II
Dependence of [3H]Ryanodine Binding to MHS and Normal SR Stimulatory
Inhibitory
Ca”’ Normal
MHS cd: Ca’+ only” Ca2+ plus 5 mM caffeine’ Ca*+ plus 5 mM AMPPNPd
ET AL.
0.34 * 0.05 0.049 +- 0.005b 0.27 f 0.02'
a As reported by Mickelson, et al. (8). b Significantly different from normal values, P < 0.05. ’ Means ? SE, calculated from the data obtained for each individual d Means + SE, calculated from the data obtained for each individual
Normal
MHS c&
(Md
0.54 * 0.12 0.070 It 0.010 0.41 kO.06
preparation preparation
utilized in Fig. 3. utilized in Fig. 4.
Ca2+
52 t 10b 22* 2 238-t36*
(GM) 1814
15 *4 74t8
SARCOPLASMIC 5 ,
RETICULUM I
q
N
n
MHS
binding to MHS (m) and FIG. 5. Effect ofcaffeine on [“Hlryanodine normal (0) SR. [“Hlryanodine binding was performed at a Ca” concentration of 1 PM, and a ryanodine concentration of 10 nM, as described under Experimental Procedures. Symbols represent means i SE of values determined for three different MHS or normal SR preparations.
As previously noted by Pessah et al. (31), the apparent Kd of the ryanodine receptor for ryanodine depended upon the components present in the binding media (Figs. 1 and 2, Table I). The Kd observed in 6 PM Ca”+ was greater than that observed in the presence of yM Ca2’ plus 10 mM caffeine, which in turn was greater than that in the presence of 6 PM Ca”+ plus 10 mM ATP. This order of increasing ability of these agents to increase the apparent affinity for ryanodine correlates with their ability to increase the SR calcium channel open state
2,
RYANODINE
255
RECEPTOR
probability (23-27, 32); i.e., the experimentally measured affinity of the receptor for ryanodine is dependent upon the open state probability of the channel. Although the kinetics of [3H]ryanodine binding to its receptor (31, 33) and its effect on calcium channel activity are complex (14-16,20-22,25), these results are consistent with those of previous studies suggesting that ryanodine associates with its receptor when the channel is in the open state (20-22, 31). In all three conditions examined, the apparent Kd of the MHS SR receptor for ryanodine was significantly less than that for normal SR, although in the presence of Ca’?+ plus ATP the magnitude of the difference was quite small (Table I). Thus, when the calcium release channel is maximally activated (P,, of approximately 1 (24)), the apparent affinities of the MHS and normal SR for [“Hlryanodine are essentially identical; only when the channel is partially active (i.e., PO of less than 1) are differences in the apparent affinity of the MHS and normal ryanodine receptor for [3H]ryanodine readily detectable. The L,, of the porcine ryanodine receptor did not vary significantly under the different incubation conditions and was similar for both MHS and normal SR (Table I). This confirms our earlier observation that the calcium release channel protein content is not significantly different in MHS and normal SR (8,9). Thus, a greater MHS SR channel content cannot explain the reports of an increased rate of MHS SR calcium efflux (3, 4, 79). Rather, differences in the regulation of the MHS SR channel open state, allowing a greater P, or calcium conductance, are more likely responsible for increased rates of MHS SR calcium release. The Ca’+ dependence of [“Hlryanodine binding to both MHS and normal SR in the presence of 5 mM
I q
N
n
MHS
6
[Ruthenium Red] (M)
[Ruthenium Red] (M)
binding to MHS (D) and normal (0) SR. [“Hlryanodine binding was performed at a Ca” FIG. 6. Effect of ruthenium red on [“Hlryanodine concentration of 6 pM, and a ryanodine concentration of 10 nM, in the absence (A) or presence (B) of 10 mM ATP. Symbols represent means t SE of values determined for four different MHS or normal SR preparations.
256
MICKELSON
4
2
0 2
[Magnesium] (mM) FIG. 7. EfIect of Mg*+ on [“Hlryanodine binding to MHS (a) and normal (0) SR. [“H]Ryanodine binding was performed at a Ca2+ concentration of 6 pM and a ryanodine concentration of 100 nM, as described under Experimental Procedures. Symbols represent means f SE of values determined for four different MHS or normal SR preparations.
caffeine or 5 mM AMPPNP exhibited a maxima in the PM Ca’+ range (Figs. 3 and 4). At any given Ca2+ concentration [“Hlryanodine binding to MHS SR was greater than to normal SR, likely due to the lower apparent Kd of the MHS SR receptor for ryanodine (Table I). The Caii for the stimulation and inhibition of SR [“HJryanodine binding varied depending on the components present in the binding media (Table II, see also (31,34)). Whereas AMPPNP did not alter the Cai,: for stimulation of [“Hlryanodine binding, caffeine significantly lowered this value. In contrast, the Caii for the inhibition of normal SR [“Hlryanodine binding was significantly increased by AMPPNP, but unaffected by caffeine. Thus, the effect of adenine nucleotide in raising the Cai,; for inhibition of [“Hlryanodine binding may be explained by AMPPNP inhibiting the channel Ca’-dependent inactivation mechanism since ryanodine appears to bind to the open state of the channel. In contrast, the lowering of the Cai: for stimulation of [3H]ryanodine binding by caffeine may be explained by caffeine stimulating the channel Ca” dependent activation mechanism. While there was an approximately 1.5-fold difference between MHS and normal SR in the Ca$: for activation of binding in the presence of Ca’+, Ca2+ plus AMPPNP, or Ca2+ plus caffeine (Table II), this was less significant than the up to 3-fold difference in the Cai; for the inhibition of binding in the presence of Ca2+, or Ca”’ plus AMPPNP. The major difference between MHS and normal SR [“Hlryanodine binding at high Ca”+ may be related to the abnormal kinetics of channel inactivation we have observed when this channel is inserted in planar lipid
ET AL
bilayers (26). In these experiments MHS channels failed to inactivate in millimolar Ca2+ concentrations (either cis or tram), resulting in a greater POfor the MHS channel (26). In the presence of PM Ca2+, caffeine stimulated, while ruthenium red and Mg” inhibited, [3H]ryanodine binding to the porcine SR (Figs. 5-7, Table III); this correlates with the ability of these agents to stimulate and inhibit respectively, both SR calcium release and single channel activity (23-25, 28, 32, 35). Interestingly, the sensitivity of SR [“Hlryanodine binding to ruthenium red inhibition was decreased in the presence of the stimulatory agent ATP (Table III). When the effects of these agents on MHS and normal SR [3H]ryanodine binding were compared it was found that less caffeine was required to stimulate, and more ruthenium red or Mg2+ was required to inhibit [3H]ryanodine binding to MHS SR than to normal SR. The small but significant difference in C0,6 for caffeine stimulation of [“Hlryanodine binding between MHS and normal SR correlates with the small difference we have observed in the C,, for caffeine stimulation of calcium release from MHS and normal SR vesicles (9). In all cases, however, there was at most a twofold difference in the C,, values for effects of caffeine, ruthenium red, and Mg2+ on [3H]ryanodine binding; the Cai: values for inhibition of [“Hlryanodine binding (Table II) and the Kd for ryanodine in 6 PM Ca”+ (Table I) demonstrated the largest difference in any value between MHS and normal SR. We conclude that the altered Ca2+ regulation of the calcium release channel (3-9,26,30) may be the primary
TABLE Sensitivity of MHS to Stimulation
III
and Normal SR t3H]Ryanodine Binding or Inhibition by Various Agents Concentration for half-maximal effect (C,,,) MHS
Agent Caffeine (mM) (+l pM Ca”‘)” Ruthenium red (PM) (+6 gM Ca”‘)’ Ruthenium red (PM) (+lO mM ATP) (+6 pM Cay’)’ Mg” (mM) (t6 fiM CZL”)~
1.7
t0.3*
Normal 3.4
* 0.2
0.079 f 0.009
0.083 f 0.011
1.9 kO.2” 0.34 * o.ofJ*
1.2 + 0.1 0.21 + 0.01
UMeans 2 SE for stimulation of [“Hlryanodine binding, calculated from the data obtained for each individual preparation utilized in Figure 5. ’ Significantly ditferent from normal values, P < 0.05. ’ Means 2 SE for inhibition of 13H]ryanodine binding, calculated from the data obtained for each individual preparation utilized in Fig. 6. “Means k SE for inhibition of [“Hlryanodine binding, calculated from the data obtained for each individual preparation utilized in Fig. 7.
SARCOPLASMIC
RETICULUM
abnormality in MHS SR, which may then result in subsequent alterations in the interaction of other ligands such as caffeine and halothane with the MHS channel, both in isolated SR (5-9,30,36) and in more intact muscle preparations (l-4). A possible explanation for our results is that there is a structural alteration in the Ca” binding domain(s) that control(s) the gating properties of the MHS calcium channel protein. However, modifications in channel accessory/regulatory proteins, including a decreased extent of phosphorylation of a 60kDa protein in MHS SR believed to play a role in regulating calcium release (37), are still possible. Definition of the molecular basis of the alterations in the MHS muscle ryanodine receptor, as well as the dihydropyridine receptor (38), should provide insight into both the mechanism of excitation-contraction coupling, and the diagnosis and treatment of this significant muscle disorder. ACKNOWLEDGMENTS \Ve thank Drs. Esther Gallant and ,Jim Ervasti for helpful discussion and Dr. William Rempel for the supply of experimental animals.
RYANODINE
257
RECEPTOR
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