Factors Influencing [3H]Ryanodine Binding to the Skeletal Muscle Ca2+Release Channel

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

248, 173–179 (1997)

AB972125

Factors Influencing [3H]Ryanodine Binding to the Skeletal Muscle Ca2/ Release Channel1 Dolores H. Needleman and Susan L. Hamilton2 The Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

Received January 16, 1997

Optimal [3H]ryanodine binding to skeletal muscle sarcoplasmic reticulum membranes is dependent on a number of factors such as Ca2/ concentration, ionic strength, and the presence of modulators of the Ca2/ release channel. The rate of association of [3H]ryanodine with its binding site is slower than a diffusion limited process, and often the binding reaches a peak value which is followed by a slow decline. This phenomenon makes it extremely difficult to determine kinetic constants for [3H]ryanodine binding. The inclusion of bovine serum albumin (BSA) or the detergent 3 - [(3 - cholamidopropyl)dimethylammonio] - 1 - propanesulfonate (Chaps) in the incubation buffer prevents the decrease in [3H]ryanodine binding observed in association studies. BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component. Pretreatment of the membranes with Chaps does not prevent the decrease in [3H]ryanodine binding, suggesting that Chaps is not exerting its effect by extracting a lipid or peripheral membrane protein. The decrease in affinity observed in the absence of BSA and Chaps appears to require the occupation of the high-affinity ryanodine binding site. Incubation for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. These combined results suggest that Chaps and BSA stabilize the ryanodine-modified Ca2/ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity, thus allowing for a more quantitative interpretation of binding data. q 1997 Academic Press

The Ca2/ release channel in the terminal cisternae of skeletal muscle releases Ca2/ from the lumen of the sarcoplasmic reticulum (SR)3 in response to a signal from the transverse tubules (t-tubules; 1). This channel is a large protein composed of four identical 565-kDa subunits and contains the binding sites for the plant alkaloid ryanodine (1–3). The binding of [3H]ryanodine is dependent upon the functional state of the channel and is used to analyze and monitor the effects of modulators of Ca2/ release channel function (4–7). The activity of the channel is modulated by many agents including caffeine, Mg 2/, ruthenium red, adenine nucleotides, sphingosine, and Ca2/, and these agents also alter [3H]ryanodine binding (4–10). In addition, several proteins are reported to interact with the Ca2/ release channel (11–17). These include aldolase (11), glyceraldehyde-3-phosphate dehydrogenase (11), triadin (12), calmodulin (13, 14), and the FK506 binding protein (FKBP12:15,16). Calcineurin, a calmodulin-dependent protein phosphatase, may also be associated with the Ca2/ release channel, possibly via an interaction with FKBP12 (17). Optimal [3H]ryanodine binding to skeletal muscle SR membranes is dependent on a number of factors such as Ca2/ concentration, ionic strength, and pH (4–7). Depending upon the incubation conditions, the affinity of [3H]ryanodine for its high-affinity site ranges from low nM to several hundred nM (4–7). The rate at which [3H]ryanodine associates with the Ca2/ release channel is extremely slow and the long incubation times needed to reach equilibrium can lead to decreased [3H]ryanodine binding, suggesting breakdown or turnover of the receptor within isolated SR membranes (18). The rate at which this decline in binding occurs is depen-

1 This work is supported by grants from the Muscular Dystrophy Association and NIH (AR41802) to S.L.H. and by a grant from the American Heart Association, Texas Affiliate (96G-1196) to D.H.N. 2 To whom correspondence should be addressed. Fax: 713-7983475.

3 Abbreviations used: SR, sarcoplasmic reticulum; t-tubules, transverse tubules; BSA, bovine serum albumin; Chaps, 3-[(3-cholamido-propyl)dimethylammonio]-1-propanesulfonate; Mops, 3-[Nmorpholino]-propanesulfonic acid; AMP-PCP, b,g-methyleneadenosine 5*-triphosphate.

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dent on the incubation temperature: an increasing rate of decline with increasing temperatures (18). This decline in [3H]ryanodine binding makes it difficult to define equilibrium conditions. Binding assays in different laboratories are performed at different temperatures with assays done at 377C for 2–3 h (19), at room temperature for 15 h (4, 5), and at 127C for 20 h (20). Obviously, the different rates of decline in [3H]ryanodine binding which occurs under these various conditions can greatly influence the interpretation of the data. Some agents, such as bovine serum albumin (BSA), appear to stabilize [3H]ryanodine binding (20, 24). Previously, our assays were performed in the presence of 100 mg/ml BSA. We have recently found that 0.1% 3[(3 - cholamidopropyl)dimethylammonio] - 1 - propanesulfonate (Chaps) is more effective than BSA for stabilizing [3H]ryanodine binding. In the present study, we characterized the effect of BSA and Chaps on [3H]ryanodine binding and developed conditions for [3H]ryanodine binding assays to prevent the gradual decline in binding which occurs with time. This technique should allow for a more quantitative interpretation of binding data. MATERIALS AND METHODS

[9,21-3H(N)]ryanodine (68 Ci/mmol) was obtained from Dupont-New England Nuclear (Boston, MA). BSA (fraction V, ú98% pure) was obtained from JRH Biosciences (Lenexa, KS). 3-[N-morpholino]propanesulfonic acid (Mops), Chaps, b,g-methyleneadenosine 5*-triphosphate (AMP-PCP), KCl, and protease inhibitors were obtained from Sigma Chemical Company (St. Louis, MO). Sarcoplasmic reticulum (SR) membrane preparation. SR membranes were prepared from rabbit skeletal muscle as previously described (21, 22). Protein concentrations were estimated following the method of Lowry et al. and used BSA in the standard curve (23). Equilibrium [3H]ryanodine binding assays. [3H]Ryanodine was incubated overnight (15–16 h) at room temperature with 10 mg SR membranes in 200 ml of buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), and 100 mM CaCl2 . Protease inhibitors were included at the following concentrations: 100 mM phenylmethylsulfonyl fluoride, 200 mM aminobenzamidine, 1 mg/ml each of aprotinin, leupeptin, and pepstatin A. [3H]Ryanodine was used in the concentration range of 1.56 to 50 nM. BSA (0.1 mg/ml) and/or 0.1% Chaps were included in the incubation mixture as indicated in the figure legends. Nonspecific binding was defined in the presence of 10 mM unlabeled ryanodine. The incubation was terminated by filtration of the sample volume through a Millipore vacuum filtration apparatus equipped with Whatman GF/F glass fiber filters, and the filters were

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washed with 5 1 3 ml of ice-cold buffer containing 0.3 KCl, 10 mM Mops (pH 7.4), and 100 mM CaCl2 . The filters were placed in scintillation vials and 5 ml Ultima Gold (Packard Instrument Co., Meriden, CT) was added to the samples. The samples were shaken for 1 h, and the amount of radioactivity was quantitated by liquid scintillation counting. [3H]Ryanodine dissociation kinetics. SR membranes (2.2 mg/ml final protein concentration) and 41 nM [3H]ryanodine were incubated for 15 h at 237C in binding buffer containing 0.3 M KCl, 0.1 mg/ml BSA, 50 mM Mops (pH 7.4), 100 mM CaCl2 , and protease inhibitors in a total volume of 600 ml. Dissociation was initiated by diluting the [3H]ryanodine-bound membranes 1:400 in binding buffer. BSA (0.1 mg/ml) or Chaps (0.1%) was also included in the binding buffer as indicated in the figure legends. At the indicated times, 400-ml aliquots were filtered, washed, and processed for scintillation counting as previously described. [3H]Ryanodine association kinetics. The association experiments were initiated by adding SR membranes (0.015–0.03 mg/ml final protein concentration) to a buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , 20 nM [3H]ryanodine, 1 mM AMP-PCP, and protease inhibitors in a total volume of 3 ml. BSA (0.1 mg/ml) and or 0.1% Chaps were included in the binding buffer as indicated in the figure legends. AMPPCP was included in the buffer to increase the association rate of [3H]ryanodine and shorten the time of assay. In the absence of AMP-PCP, the association rate of [3H]ryanodine is extremely slow and, at low ligand concentrations, requires long incubation times. At the indicated times, 100-ml aliquots were filtered, washed, and processed for scintillation as previously described. Data analysis. Dissociation and association data were fit by nonlinear curve fitting routines using Sigma Plot (Jandel Scientific, Corte Madera, CA) or Deltagraph Professional (Corte Madera, CA). Results are presented as { standard error of the mean (SEM). M

RESULTS

BSA and Chaps enhance [3H]ryanodine binding. The apparent affinity of SR membranes for [3H]ryanodine is dependent on the conditions of the binding assay. We have previously shown that the apparent KD for binding is a weighted average of the Kd’s for binding to different functional states of the channel (5). In addition to the effect of known modulators of the Ca2/ release channel on the binding of [3H]ryanodine, some agents, such as BSA, appear to stabilize [3H]ryanodine binding (20, 24). Previously, our assays were performed in the presence of 100 mg/ml BSA. We have recently found that 0.1% Chaps is more effective than BSA for stabilizing [3H]ryanodine binding. To explore this sta-

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FIG. 1. Scatchard analysis of [3H]ryanodine binding in the presence and absence of BSA and Chaps. SR membranes (10 mg) were incubated overnight (15 h) at room temperature (237C) with 1.56 to 50 nM [3H]ryanodine in 200 ml binding buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , and protease inhibitors. Nonspecific binding was defined in the presence of 10 mM unlabeled ryanodine. Filtration and scintillation counting were performed as described under Materials and Methods. The binding curves were fitted by linear regression. The Kd and Bmax for the control curve (l) were 17.7 nM and 17.8 pmol/mg, respectively. The Kd and Bmax values in the presence of 0.1 mg/ml BSA (s) were 13.2 nM and 17 pmol/mg, respectively. The Kd and Bmax values in the presence of 0.1% Chaps (j) were 4.8 nM and 17 pmol/mg, respectively. The Kd and Bmax values in the presence of both 0.1 mg/ml BSA and 0.1% Chaps (h) were 5.7 nM and 17 pmol/mg, respectively.

bilization phenomenon, we further characterized the effect of BSA and Chaps on [3H]ryanodine binding. As seen in Fig. 1, the presence of 0.1 mg/ml BSA and/ or 0.1% Chaps increased the apparent affinity of the membranes for [3H]ryanodine, but had no significant effect on the number of binding sites. The KD shifted from 23.4 { 2.3 nM in the control incubations to 17.1 { 1.8 nM (0.01 õ P õ 0.05, paired t test) and 5.3 { 0.7 nM (P õ 0.01, paired t test) in the presence of 0.1 mg/ ml BSA and 0.1% Chaps, respectively (n Å 3). The addition of BSA and Chaps together to the incubation buffer decreased the KD to 6.0 { 0.9 nM (n Å 3, P õ 0.01, paired t test). In addition to Chaps and BSA, other agents increase the affinity of the membranes for [3H]ryanodine. These agents include g-globulin, aldolase, nonfat dried milk (Bio-Rad blocker), and fatty acid-free BSA (data not shown). This increase in [3H]ryanodine binding by Chaps and BSA could be accomplished by preventing (i) degradation of the ligand, (ii) degradation or denaturation of protein, or (iii) enzymatic modification of the Ca2/ release channel. Incubation of [3H]ryanodine in buffer for either t Å 0 h or t Å 15 h at room temperature prior to the initiation of association by the addition of membranes produces identical curves, suggesting

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that breakdown of the ligand is not occurring (data not shown). To determine if proteolytic degradation is the cause of the change in affinity of [3H]ryanodine, we examined the effect of incubation of membranes on the endogenous proteolysis of the 565-kDa subunit of the Ca2/ release channel by membrane-associated proteases. The Ca2/ release channel is a major substrate for the Ca2/-activated neutral protease, calpain (25–27). Endogenous proteolysis by calpain sequentially cleaves the Ca2/ release channel with initial cleavage of the 565-kDa subunit into 410- and 160-kDa peptides (25, 26). The physiological significance of this cleavage is unknown. In the current studies, SR membranes were incubated under binding conditions for 0 and 500 min at room temperature in the presence and absence of either 0.1 mg/ml BSA or 0.1% Chaps. The membrane proteins were separated using SDS–PAGE, and the amount of Coomassie-stained protein was quantitated by densitometry using a Pharmacia ImageMaster densitometer and the Image Master software package. The degree of endogenous proteolysis was estimated by quantitating the ratio of the 565 kDa/410 kDa bands (Fig. 2). Addition of either BSA or Chaps to the membranes had no significant effect on the ratio of the 565 kDa/410 kDa bands. The effect of BSA and Chaps on [3H]ryanodine binding is apparently not due to the ability of these reagents to slow proteolysis of the Ca2/ release channel. Association of [3H]ryanodine with SR membranes is characterized by a rapid rise to peak followed by a slow decline. In the presence of AMP-PCP, the association of [3H]ryanodine with SR membranes is characterized by an initial rapid increase in binding followed by a gradual decline (Fig. 3A). A similar loss of binding with time was observed by Carroll et al. (18). The actual onset time of the gradual decline in binding varies among membrane preparations. In the presence of 1 mM AMP-PCP, maximal binding is reached between 50 and 200 min and a decline in [3H]ryanodine binding is first detected after 70–300 min. Although the ‘‘overshoot’’ phenomenon was not seen in the absence of AMP-PCP, the amount of binding reached at equilibrium was significantly less in the absence of BSA and Chaps (Fig. 3B). The ‘‘overshoot’’ is probably not seen because the association rate of [3H]ryanodine with the membranes is slow compared to the onset of the process that leads to a decrease in binding. Therefore, in the absence of BSA or Chaps, the association curve can have a deceptively normal appearance since the rapid onset of the process which decreases binding is masked. To determine if the addition of either BSA or Chaps can reverse the slow decline in [3H]ryanodine binding, we added these reagents to the incubation buffer at various times after the addition of [3H]ryanodine (n Å

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FIG. 2. Effect of BSA and Chaps on the endogenous proteolysis of the Ca2/ release channel. SR membranes (0.1 mg/ml) were incubated in 3 ml buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , and 5 nM ryanodine in absence or presence of either 0.1 mg/ ml BSA or 0.1% Chaps. At t Å 0 min and t Å 500 min, the membranes were pelleted by centrifugation in a Ti70.1 rotor at 35,000 rpm for 35 min. The membranes were resuspended in 100 ml buffer. The membrane proteins were subsequently separated by SDS–PAGE (4.5% gel) and the amount of coomassie-stained protein was quantitated by densitometry using a Pharmacia ImageMaster densitometer and the Image Master software package. The degree of endogenous proteolysis was estimated by quantitating the ratio of the 565 kDa/ 410 kDa bands. Error bars represent the SEM of n Å 3 replicate samples.

3). A representative experiment examining the effect of addition of 0.1% Chaps at different times is shown in Fig. 4. As seen previously, there was a rapid increase in [3H]ryanodine binding which reached a peak and then declined. Addition of Chaps at t Å 0 prevented the decline in [3H]ryanodine binding observed after 100 min and increased maximal binding. Addition of CHAPS at t Å 100, 200, 265, and 400 min prevented subsequent loss of [3H]ryanodine binding and partially restored binding. However, the extent of the recovery of binding decreases with the time of addition of Chaps. The addition of BSA at different times gave similar results (data not shown). These data indicate that part of the decline in [3H]ryanodine binding is irreversible. Chaps and BSA could be stabilizing [3H]ryanodine binding (i) by preventing denaturation, (ii) by promoting renaturation, (iii) by inhibiting the action of an enzyme, or (iv) by extracting an endogenous modulator of ryanodine binding from the SR membranes. To eliminate the last possibility, the effect of pretreatment of SR membranes with Chaps on the binding of [3H]-

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ryanodine to its high-affinity site was examined. In this experiment, SR membranes were incubated in the presence 0.1% Chaps and then washed by pelleting prior to the association experiment (n Å 4). A representative experiment is shown in Fig. 5. Even after the Chaps wash, the association of [3H]ryanodine is characterized by an overshoot in binding, and the decline in binding can be prevented by the presence of Chaps in the association buffer. The decrease in maximal [3H]ryanodine binding observed upon Chaps pretreatment is due to loss of protein upon pelleting and resuspension. Chaps does not appear to be extracting an endogenous modulator of [3H]ryanodine binding, but rather is preventing an alteration in the protein which leads to a lower affinity for [3H]ryanodine. If this decline is due to the action of an enzyme, Chaps and BSA do not extract the enzyme but rather appear to protect the Ca2/ release channel from its action. Since the decline in [3H]ryanodine binding is due to a change in affinity rather than a loss in the number of binding sites, the alteration which results in decreased [3H]ryanodine binding may only be a local change in the binding site. The binding of ryanodine to its high-affinity site causes the Ca2/ release channel to adopt a different conformational state which is characterized by long openings and a reduced channel conductance (2, 4, 6, 7, 9). To determine if the events which lead to a decline in ryanodine binding required the conformational state adopted by the ryanodine-modified channel, we examined the effect of extended incubation of SR membranes prior to the initiation of association by the addition of [3H]ryanodine (Fig. 6). In this experiment, SR membranes were either freshly diluted into association buffer or allowed to incubate in buffer for 15 h prior to the addition of [3H]ryanodine. An overshoot in the association curve was observed in both cases. Incubation of SR membranes in buffer prior to the initiation of association did not lead to a decrease in the amount [3H]ryanodine binding compared to control curves (n Å 5) and occasionally produced slightly higher levels of maximal binding (Fig. 6). These findings suggest that the decrease in affinity of membranes for [3H]ryanodine requires the occupation of the site by ryanodine. Either the ryanodine-bound channel is less stable or it is more susceptible to the action of an enzyme. The decrease in binding over time can be partially explained by an increase in the rate of dissociation of [3H]ryanodine. We examined the effect of BSA and Chaps on the dissociation of bound [3H]ryanodine from SR membranes to determine the effect of these reagents on dissociation kinetics. The dissociation of bound [3H]ryanodine from SR membranes is slower in the presence of either 0.1 mg/ml BSA or 0.1% Chaps than in their absence (Fig. 7). In the absence of these agents, the dissociation of bound ryanodine is charac-

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FIG. 3. Association of [3H]ryanodine to rabbit skeletal muscle SR membranes. (A) SR membranes (90 mg) were added to 3 ml of 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , 20 nM [3H]ryanodine, 1 mM AMP-PCP, protease inhibitors, and no additions (l), 0.1 mg/ml (1.2 mM) BSA (s), or 0.1% Chaps (j). At the indicated times, 100-ml aliquots were filtered. Filtration and scintillation counting were performed as described under Materials and Methods. (B) Effect of Chaps and BSA on association kinetics in the absence of AMP-PCP in the buffer. The [3H]ryanodine concentration was 14 nM and the protein concentration was 0.06 mg/ml. No additions (l); 0.1 mg/ml (1.2 mM) BSA and 0.1% Chaps (s). Data in A and B were obtained with different membrane preparations.

terized by two components, a fast component (koff Å 0.015 min01) and a slow component (koff Å 0.0025 min01). In the presence of either 0.1% Chaps or 0.1 mg/ ml BSA, the relative amount of the fast component is decreased. In the absence of BSA or Chaps, the relative amount of the fast component of dissociation is 69.2 { 14.5% (n Å 4) of the dissociation. The relative amount of the fast component decreases to 22.0 { 8.3% (n Å 3)

FIG. 4. Effect of Chaps on the decrease in [3H]ryanodine binding over time. SR membranes (180 mg) were added to 6 ml of 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , 20 nM [3H]ryanodine, 1 mM AMP-PCP, and protease inhibitors. At t Å 0, 100, 200, 265, and 400 min, Chaps (0.1% final) was added to the incubation buffer. Aliquots (200 ml) were filtered at the indicated times. Filtration and scintillation counting were performed as described under Materials and Methods. Control curve (l), Chaps (s).

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and 17.9 { 3.0% (n Å 3) in the presence of BSA and Chaps, respectively. The alteration in the apparent affinity of [3H]ryanodine for the high-affinity site appears to be due partially to a change in the rate of dissociation of [3H]ryanodine from the binding site. DISCUSSION

[3H]Ryanodine binding can be used to monitor changes in the functional state of the Ca2/ release channel (4–7). [3H]Ryanodine binding to SR membranes is both time and temperature dependent (18). At room temperature, the rate at which ryanodine binding reaches equilibrium is extremely slow and the long incubation times can lead to decreased [3H]ryanodine binding, suggesting breakdown or denaturation of the receptor in isolated SR membranes (18). Increasing the incubation temperature to 377C greatly shortens the time required to reach maximal binding, but also promotes the rate of decrease in binding, thus making it difficult to define equilibrium conditions. [3H]Ryanodine binding assays in different laboratories are performed using different temperatures and times (4, 5, 18–20). The decline in [3H]ryanodine binding which occurs under these conditions can greatly influence the interpretation of data. In the present study, we report that 0.1 mg/ml (1.2 mM) BSA and 0.1% Chaps slows the irreversible decline in the affinity of SR membranes for [3H]ryanodine under conditions where the assays are performed at room temperature. BSA or Chaps appear to stabilize the [3H]ryanodine binding site on SR membranes and pre-

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FIG. 5. Effect of pretreatment of SR membranes with Chaps on the association of [3H]ryanodine. SR membranes (0.5 mg) were incubated in 1 ml of buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , and protease inhibitors in the presence and absence of 0.1% Chaps for 30 min. The membranes were pelleted by centrifugation for 5 min at 30 psi using a Beckman airfuge. Control (A) and Chapstreated membranes (B) were resuspended in buffer and were added to 3 ml of 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , 20 nM [3H]ryanodine, 1 mM AMP-PCP, and protease inhibitors in the presence (s) and absence (l) of 0.1% Chaps. At the indicated times, 100-ml aliquots were filtered. Filtration and scintillation counting were performed as described under Materials and Methods. The protein concentration for A and B was 0.03 and 0.015 mg/ml, respectively.

vent the decrease in [3H]ryanodine binding observed in association studies. Addition of either BSA or Chaps slows this decline in binding partially by preventing a conversion to a more rapidly dissociating component of the dissociation. Pretreatment of the membranes with Chaps does not prevent the decrease in affinity. The decrease in affinity observed in the absence of BSA and Chaps in the assay buffer appears to require the

FIG. 6. Effect of preincubation time on the association of [3H]ryanodine to SR membranes. SR membranes (120 mg) were incubated for t Å 0 hr (l) and 15 h (s) at room temperature in 2.2 ml buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , 1 mM AMP-PCP, and protease inhibitors prior to the initiation of association by the addition of 20 nM [3H]Ryanodine. Aliquots (200 ml) were filtered at the indicated times. Filtration and scintillation counting were performed as described under Materials and Methods.

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occupation of the high-affinity ryanodine binding site. Incubation in buffer for extended times in the absence of [3H]ryanodine prior to the initiation of the association produced similar curves to those obtained without preincubation. If the decline were independent of occupation of the site by [3H]ryanodine, the events which produced the decline should have occurred during the

FIG. 7. Dissociation of bound [3H]ryanodine from rabbit skeletal muscle SR membranes. SR membranes were incubated overnight (15 h) at room temperature (23 7C) with 31 nM [3H]ryanodine in 400 ml binding buffer containing 0.3 M KCl, 50 mM Mops (pH 7.4), 100 mM CaCl2 , and protease inhibitors. At t Å 0, 40 ml membranes was diluted into 16 ml binding buffer containing no additions (l), 0.1 mg/ ml BSA (s), and 0.1% Chaps (h). At the indicated times, 400-ml aliquots were filtered. Filtration and scintillation counting were performed as described under Materials and Methods.

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preincubation. The curves were essentially identical with and without preincubation of the membranes. These results indicate that the ryanodine-modified channel is more susceptible to the alteration in affinity. These combined results suggest that 0.1% Chaps and 0.1 mg/ml (1.2 mM) BSA stabilize the ryanodine-modified Ca2/ release channel by preventing an alteration in the ryanodine binding site which leads to decreased affinity. Lu et al. reported that 10 mM BSA increased the number of binding sites without changing affinity of [3H]ryanodine for SR membranes (20). We have, however, never detected a Bmax change with BSA using the SR protein concentrations described under Materials and Methods. The alteration in the ryanodine-modified Ca2/ release channel may reflect either a partial denaturation in the protein or a change in protein structure due to the action of an enzyme. Detergents, liposomes, and detergent/lipid micelles have been used to facilitate the renaturation of proteins (28–30). It is conceivable that Chaps, a detergent, and BSA, a lipid-binding protein (31), either prevent denaturation or assist in the local refolding of the ryanodine receptor. Alternatively, Chaps could be inhibiting the action of an enzyme while BSA serves as an alternate substrate for this enzymatic activity. Proteins known to interact with the Ca2/ release channel such as aldolase (11), glyceraldehyde-3phosphate dehydrogenase (11), triadin (12), calmodulin (13, 14), FKBP12 (15, 16), or calcineurin (17) may play a role in the events which lead to the alteration in the ryanodine-modified Ca2/ release channel. We are currently pursuing these avenues of investigation. In summary, the inclusion of BSA or Chaps in [3H]ryanodine binding assays stabilizes binding, thus allowing for a more quantitative interpretation of binding data. ACKNOWLEDGMENTS The authors thank Barbara Williams, Fred Mandel, Bahman Aghdasi, and YiLi Wu for their helpful comments during the preparation of the manuscript. We also thank Manuel Escobar for his excellent technical assistance and Lynda Attaway for her help in preparation of the manuscript.

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