[3h]ryanodine binding sites to high affinity sites on the skeletal

THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Bidom, Inc.

Vol. 268, No. 28, Issue of October 5, pp. 20974-20982, 1993 Printed in U S A .

Relationship of Low Affinity [3H]Ryanodine Binding Sites to High Affinity Sites on the Skeletal Muscle Ca2+ Release Channel*

(Received for publication, February 1, 1993, and in revised form, July 8, 1993)

Jian Ping Wang, Dolores H. Needleman, and Susan L. Hamilton$ From the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which ['Hlryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of 13H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conver- sion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min" to one with a dissociation rate constant of less than 0.00025 min-l. The low affinity site itself has a dissociation rate constant of 0.013 min-l and dissociation from this site is unaffected by the presence of 100 p~ unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation.

Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3HlPN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.

The calcium channel in the terminal cisternae of skeletal muscle releases calcium from the lumen of the sarcoplasmic reticulum (SR)l in response to a signal from the transverse tubules (t-tubules) (1). A 450-kDa protein has been purified from skeletal muscle SR membranes and shown to be both the binding protein for the plant alkaloid, ryanodine, and the Ca2+ release channel (2-6). The binding of l3H1ryanodine to the Ca2+ release channel is dependent on the functional state of the channel (7-9) and can be used to analyze and monitor the effects of modulators of Ca2+ release channel function.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by grants from the Muscular Dystrophy Association and National Institutes of Health Grants HL- 37028, HL-37044, and AR-41729.

$ To whom correspondence should be addressed. The abbreviations used are: SR, sarcoplasmic reticulum; t-tubule,

transverse tubule; MOPS, 3-(N-morpholino)propanesulfonic acid; BSA, bovine serum albumin; AMP-PCP, adenosine 5'-(P,y-methylene)trii- phosphate.

The binding of 13Hlryanodine to the Ca2+ release channel is complex in that there are both high and low affinity binding sites for ryanodine (2, 8-16). The relationship of low affinity sites to the high affinity site is not clear. Occupation of a low affinity site by ryanodine blocks or inhibits the Ca2+ release channel (10,12,16). It has been reported that there is only one high affinity site for [3Hlryanodine per receptor (2). Binding of ryanodine to this site locks the channel into an open, low conductance state (10, 12). Since the Ca2+ release channel is thought to be a homotetramer (121, the single site either must represent a site to which each of the monomers contribute or must arise from the negatively cooperative binding of ryanodine to one of four initially identical sites.

McGrew et al. (16) reported two types of ryanodine binding in skeletal muscle, high affinity (Kd = 5-10 nM) and low affinity (Kd = 3 PM). Lai et al. (12) reported a ratio of 1:3 of high affinity (Kd = 8 nM) to low affinity (Kd = 5 PM) binding sites for L3H1ryanodine in SR membranes and reported that the relative ratio of high affinity to low affinity sites was dependent upon the concentration of free Ca2+, suggesting interconvertible sites. Reduction in free Ca2+ to <0.1 PM or trypsin diges- tion of the membranes (12, 15) resulted in the loss of high affinity but not low affinity binding. Hill plot analysis from binding data obtained at 20 PM and 1 mM Ca2+ showed nonlinear behavior over the range of nanomolar to micromolar ryanodine.

Pessah and Zimanyi (14) reported multiple ryanodine binding sites in both rabbit skeletal and rat cardiac SR membranes with dissociation constants of 1-4 n~ for the high affinity site and 30-50 nM, 500-800 nM, and 2-4 PM for the low affinity sites in both preparations. They also reported multiple Hill coefficients of less than 1 in both preparations. The existence of multiple low affinity binding sites and Hill coefficients of less than 1 was interpreted as evidence for the negatively cooperative binding of ryanodine at one of four initially identical sites. Such a mechanism requires an increase in the rate of dissociation of bound 13H]ryanodine in the presence of excess unlabeled ryanodine andor a slowing of [3Hlryanodine association with increasing occupancy. The interpretation of the kinetics of ryanodine binding is difficult. There are several reports in which a slowing of the rate of dissociation of bound [3H]ryanodine in the presence of excess unlabeled ryanodine has been observed (7, 12, 15, 16). McGrew et al. (16) interpret this slowing of the dissociation rate in terms of positive cooperativity. Buck et al. (13) recently reported observing both an increase in the rate of dissociation of bound [3Hlryanodine and a slowing in the rate of association of l3H1ryanodine as a function of occupancy.

In the present study, we examined the specificity of low affinity ryanodine binding sites and we analyzed the association and dissociation kinetics of l3H1ryanodine binding at concentrations above that required to occupy fully the high affinity site. We propose a model for the interaction between high and low affinity binding sites on the Ca2+ release channel, which is

20974

Low Affinity Ryanodine Binding Sites 20975

consistent with the equilibrium and kinetic [ 3 H l ~ a n ~ i n e binding data.

EXPERIMENTAL PROCEDURES

Materials

[9,21-3H]Ryanodine (61.5 Cilmmol) was purchased from Du Pont- New England Nuclear. (+)-[methyZ-3HlPN200-110 (80 Cilmmol) was purchased from Amersham Corp. Unlabeled nitrendipine was supplied by Miles Laboratories. Ryanodine was from Calbiochem. Neomycin sul- fate, ruthenium red, and MOPS were obtained from Sigma.

Sarcoplasmic Reticulum Membrane Preparation

SR membranes were prepared from rabbit skeletal muscle and were fractionated using sucrose gradient centrifugation (17, 18).

Purification of Ryanodine Receptor

Rabbit skeletal muscle ryanodine receptor was purified as previously described (5).

Equilibrium L3HIRyanodine Binding

[3H]Ryanodine was incubated overnight (15-17 h) at room temperature (23 "C) with 5-50 pg of SR membranes in 10&250 pl of buffer containing 300 mM KCI, 20 mM MOPS (pH 7.20), 100 pg/ml bovine serum albumin (BSA), and 100 PM Ca2+. Protease inhibitors were in- cluded in all steps at the following concentrations: 100 PM phenylmeth- ylsulfonyl fluoride, 200 p~ aminobenzamidine, 1 pg/ml each of aproti- nin, leupeptin, and pepstatin A. [3Hlryanodine was used in the concentration range of 0.39 nM to 10 PM and was diluted with unlabeled ryanodine as indicated in the figure legends. Nonspecific binding was defined in the presence of either 10 or 100 PM ryanodine as indicated in the figure legends. The incubation was ended by rapid filtration of the entire sample volume through Whatman GF/F glass fiber filters and washed five times with 5 ml of ice-cold wash buffer containing 0.3 M KCl, 100 p~ Ca2*, and 10 mM MOPS (pH 7.2). The filters were placed in scintillation vials, and 5 ml of Ready Proteinm (Beckman Instruments) were added to the vials. The vials were capped, and the samples were shaken for 1 h at room temperature (23 "C). The radioactivity bound to the filters was quantitated by liquid scintillation counting.

The binding of [3Hlryanodine to purified receptor was assayed in 200 pl of buffer (1 mM AMP-PCP, 520 p~ CaCI,, 100 pg/ml BSA, 1 M KCI, 50 mM MOPS (pH 7.4)) by incubating [3Hlryanodine (4.8 nM to 5 PM diluted

0 t J 6 2 b e 4

0 I-

z t -

0 500 1000 1500

TIME (minu tes ) FIG. 1. Proteolysis of the 460-kDa band is not detected during

the time co- of the experiments. To determine if proteolysis is a significant problem in the course of the 15-h equilibrium binding stud- ies, we examined the effect of incubation time on the polypeptide com- position by SDS-polyacrylamide gel electrophoresis. Parallel samples

the presence of 100 pg/ml BSA and protease inhibitors as previously (45 mg/ml) were incubated on ice and at room temperature (23 "C) in

described. At the time indicated, the membranes were pelleted by cen- trifuging in a Beckman Airfuge for 3 min at 30 p.8.i. The pellets were resuspended in SDS sample buffer and frozen at -80 "C until electrophoresis. The optical density of the 450-kDa band is plotted as percent of the total proteins in the lane. The gels were scanned using a Phar- macia Image Master densitometer. 0, W, two separate determinations at room temperature (23 "C). 0.0, two separate determinations at 4 "C.

1:75 with unlabeled ryanodine) with 2 pg of protein for 6 h at room temperature (23 "C). Soybean lecithin (20 mglml suspension in assay buffer) was added to purified receptor for a final concentration of 10 mg/ml prior to the addition of protein to the assay. The assay was terminated by the addition of 100 pl of a solution containing 5 mdml of both BSA and rabbit y-globulin followed by 5 ml of 10% polyethylene glycol (M, 8000) in ice-cold wash buffer (0.3 M KC1,lOO PM Ca2', 10 mM MOPS (pH 7.4)). After 15 min on ice, the assay was filtered on Whatman GF/F filters followed by three 5-ml washes of the 10% polyethylene glycol solution. The amount of radioactivity bound to the filters was quantitated as previously described.

[3H]Ryanodine Dissociation Kinetics

SR membranes (0.4-0.5 mg of protein with 5-30 pmoYmg binding sites) were equilibrated for 15 h at room temperature (23 "C) with [3H]ryanodine at the indicated concentrations in 0.3 M KC1, 20 m~ MOPS (pH 7.20), 100 PM Ca2+, 100 pglml BSA, and protease inhibitors. The dissociation was initiated by diluting the membranes such that the final concentration of I3H1ryanodine was less than 0.05 I", and experiments were performed in both the presence and absence of 100 PM ryanodine in the dilution solution. Dissociation experiments were performed at room temperature (23 "C), and aliquots were filtered at the indicated intervals of time for a total of either 24 or 48 h.

13H]Ryanodine Association Kinetics

The association experiments were initiated by adding SR membranes (0.32 mg of protein with 3.1 pmol/mg binding sites) to a buffer containing 0.3 M KCl, 20 mM MOPS (pH 7.20), 100 p~ Ca2+, 100 pg/ml BSA, 1 mM AMP-PCP, protease inhibitors, and 0.05-5.9 nM [3Hlryanodine. At the appropriate intervals of time, 100-p1 aliquots were filtered, washed, and processed for radioactivity as previously described. Equilibrium values were obtained after 12-15 h. k,, was derived from the slope of a plot of ln(B,JB, - B) uersus time or from nonlinear curve fits using Sigma Plot (Jandel Scientific).

[3HlPN200-l10 Binding

Membranes (10 pg) were incubated with 1 nM [3H]PN200-110 in 2 ml of 50 mM MOPS (pH 7.4) for 2 h a t room temperature (23 "C) in the dark. The binding was terminated by rapid filtration through Whatman GF/F filters followed by five 5-ml washes with ice-cold distilled water. The amount of radioactivity bound to the filters was quantitated as previously described. Nonspecific binding was defined in the presence of 1 p~ nitrendipine.

Protein Determination

Protein concentrations were estimated by the method of Lowry et al. (19).

Data Analysis

Single component Scatchard plots were analyzed by using a least squares fit linear regression program. Binding curves with multiple sites, dissociation data, and association data, were fit by nonlinear curve-fitting routines using Sigma Plot (Jandel Scientific) as described below.

Equilibrium Binding Data-Equilibrium binding data (bound uersus free ligand concentration) were fit with reiterative fits by Sigma plot using the following function.

f=-+- A B

1 + - 1+- a b x x

f was fit to y , where x = free concentration of ryanodine, y = bound ligand, A = Bmaxclj, B = B,,,,,, a = Kdl , and b = Kd2.

Association-The amount of ligand bound as function of time after addition of the radioligand was fit with the following:

f = A x ( l - e x p ( - t ) ) + B X ( l - e x p ( - t ) ) (Ea. 2)

where x = time, y = total amount bound at time t, A = amount bound to component 1 at equilibrium, B = amount bound to component 2 at equilibrium, C= amount bound to component 3 at equilibrium, a =

20976 Low Affinity Ryanodine Binding Sites

f = A xexp ( -- :) + B xexp (-i) +C x exp( -f) (Eq.3)

f was fit to y, where x = time, y = total amount bound at time t , A = amount bound to component 1 at t = 0, E = amount bound to component 2 at t = 0, C = amount bound to component 3 at t = 0, a = Uk-,, b = l /k-2, and e = lJk-s.

RESULTS Equilibrium Binding of ~HIRyanodim to S R Membmnes

-The rate at which [3H]ryanodine reaches its binding site is extremely slow (7, 10,20). In the absence of BSA and protease inhibitors, the incubation times required to reach equilibrium can lead to decreased ryanodine binding (lo), suggesting break- down of the receptor. To circumvent this problem, we include

A

1.4

E 1.2

2 1.0

5 0.8

g 0.8 i a

\ 0.4 1 1 OS2 0.0 0 L_ 10 20 30

BOUND (pmole/mg)

7 C

1.s w E 2 1.0 z 3

0.1

0.0

0 C O R P O L 0 moMTcII

0 10 ao 30 i

both BSA (100 pg/ml) and a mixture of protease inhibitors in our incubation mixtures. Under these conditions, there is very little apparent proteolysis of the 450-kDa band (Fig. 1). Occa- sionally, in some membrane preparations there is a slow decrease in binding detected after 15 h. These experiments were discarded.

The model of negatively cooperative binding of ryanodine to the Ca2+ release channel predicts a ratio of one high affinity site to three low affinity sites. Scatchard analysis of 13H]ryanodine binding to SR membranes at [3H]ryanodine concentrations ranging from 0.4 n~ to 5 p~ was performed (Fig. 2) to quantitate the number of high and low affinity binding sites. Nonspecific binding was defined in the presence of 100 p ryanodine. At high concentrations of [3Hlryanodine, there is a curvature to the Scatchard plot. In 100 PM Ca2+, 300 m~ KC1, 20 m~ MOPS (pH 7.21, the apparent Kd for binding to the high

6

0 c o m a 0 1 mM “PCP

0 l u 0 I 10 1s 20 Ob

BOUND

D 2.0

1 .I

1 .I

1.4

1.2

2 1.0 z 8 0.6

0.4

0.2

0.0

=) 0.1

0 10 20 30 40 10 I D

BOUND BOUND FXO. 2. Scatchard analysis of the binding of [aH]ryanodine to rabbit skeletal muscle SR membranes. In each experiment, 50 pg of SR

membranes were incubated with PHlryanodine (diluted 1:75 with unlabeled ryanodine) over a concentration range of 0.4 md to 5 p~ in 0.3 M KC1, 100 PM free Ca2+, 20 m~ MOPS (pH 7.2), and 100 pdml BSA overnight (15-17 h) at room temperature (23 “C). Nonspecific binding was defined in the presence of 100 PM unlabeled ryanodine. Filtration and scintillation counting were performed as described under “Experimental Procedures.” Binding curves were fit using a nonlinear curve-fitting program or by linear regression of data plotted as boundfree uersus bound. A, Scatchard analysis of the binding of [3H]ryanodine to SR membranes at 100 p~ (0) and at 1 p~ (0) free Ca2+. Ca*+ was buffered to these concentrations with

= 913 nM, B,,c2, = 12 pmoVmg, and Kd2 = 1.1 p ~ . B, Scatchard analysis of the binding of [3H]ryanodine to SR membranes in the presence (0) and 1 m~ EGTA. At 100 p~ Ca2+, Bmmcl) = 18 pmoUmg, KdI = 11 n ~ , Bmmcz, = 12 pmoUmg, and Kd2 = 1.1 p ~ . At 1 p~ Ca2+, Bmmm = 19 pmoVmg, &I

absence (0) of 1 mM AMP-PCP. The fitted values for the control curve were Kdl = 8 n ~ , Bmslcl, = 10 pmoumg, Kd2 = 1 p ~ , and B,,(z) = 16 pmovmg.

of the binding of [SH]ryanodine to SR membranes in the presence (0) and absence (0) of 10 p~ neomycin. In C, &, = 9 md, Bmm(l) = 18 PrnoUmg, In the presence of AMP-PCP, the fitted values were Kdl = 1.7 I”, Brn=(,) = 12 pmoVmg, G2 = 1 PM, and Bmd2, = 11 pmoUmg. C, Scatchard analysis

Kdz = 1 PM, Bmm(2) = 20 pmoVmg for the control and Kd, = 28 nM, B,,,,, = 17 pmoVmg, KA = 1 p ~ , BmSc2, = 20 pmoUmg in the presence of neomycin. D, Scatchard analysis of the binding of [3H]ryanodine to SR membranes in 300 m~ KCl, 100 p~ free Ca2+, 50 m~ MOPS (pH 7.2) (0) or in 150 x” K2S0,, 100 p~ h e Ca2+, 50 mM MOPS (pH 7.2) (0). In D , Kdl = 7.6 md, B,,,, = 20 pmoVmg, Kd2 = 1.5 PM, Bm,(2) = 25 pmoVmg for the experiment with C1- and Kd, = 29 n ~ , Bmm(,) = 24 pmoVmg, Kd2 = 1 p ~ , Bmmcz, = 27 pmoVmg for the experiment with SO:-. Each set of Scatchard analyses was performed using a separate membrane preparation.


PURIFIED RYANODINE RECEPTOR

2o r“--

0 0 200 400 600 800 1000 1200 1400

BOUND FIG. 3. Scatchard analysis of the binding of [aHlryanodine to

purified ryanodine receptor. Purified receptor (2 pg) was incubated with [3H]ryanodine (2.4 nM to 5 p~ diluted 1:75 with unlabeled ryanodine) in 200 pl of binding buffer consisting of 1 m~ AMP-PCP, 520 PM Ca2+, 100 pg/ml BSA, 1 M KCl, 10 mglml soybean lecithin, 50 m~ MOPS (pH 7.4) for 6 h at room temperature (23 “C). Filtration and scintillation counting were performed as described under “Experimental Proce- dures.” Nonspecific binding was defined in the presence of 100 p~ unlabeled ryanodine. The Kd and B,,, values for the high affinity site are 8 n~ and 400 pmol/mg. The Kd and B,,, values for the low affinity site are 2 PM and 469 pmoL’mg.

affinity site is 8.2 2 1.8 nM (n = 5). Because the binding to the low affinity site is never saturated, it is difficult to quantitate accurately either the apparent Kd or the B,,, for binding of [3Hlryanodine to this site. Higher concentrations of L3H1ryano- dine result in a dramatic decrease in the binding, suggesting that the ligand may precipitate at high concentrations. Our data can be fit with a low affinity site with an apparent Kd of 1.1 2 0.2 p~ (n = 5). The ratio of high affinity sites to low affinity sites is 0.93 2 0.30 (n = 5).

We have examined the effects of decreased Ca2+ (Fig. 2 A ) , AMP-PCP (Fig. 2 B ) , which is a channel activator, and neomycin (Fig. 2C), which is a channel inhibitor, on the high and low affinity binding sites for [3H]ryanodine. We have also examined the effects of having SO:- instead of C1- as the counterion in the binding solution (Fig. 20 ). With either AMP-PCP or neomycin, the data can be fit (solid lines) with a change in the apparent affinity of the high affinity site with no change in either the Kd or the B,,, for the binding of [3Hlryanodine to the low affinity site. Decreasing the Ca2+ lowers the apparent affinity of the high affinity binding site to a value close to that of the low affinity site. This also occurs when SO:- is used as the counterion (data not shown), suggesting that this effect is due to the decrease in Ca2+. Likewise the effect of replacement of C1- with SO:- is consistent with a decrease in the affinity of the high affinity site with little or no effect on the low affinity site.

Low affinity binding is also detected in the purified Ca2+ release channel. Scatchard analysis of binding to the purified protein at L3H1ryanodine concentrations ranging from 2.4 nM to 5 p~ is shown in Fig. 3. In Fig. 3, the data were fit for a low affinity site with an apparent Kd for binding to the low affinity site of 2 p~ and a B,,, of 469 pmol/mg. The Kd for binding to the high affinity site is 8 nM, and the B,,, for this site is 400 pmol/mg. With these approximated B,, values, the calculated ratio of high affinity to low affinity sites is 0.85.

Dissociation of Bound PHJRyanodine-To analyze the relationship between high and low affinity binding sites, we examined the dissociation of [3H]ryanodine as a function of occupancy by either dilution alone or dilution in the presence of

excess unlabeled ligand. Earlier work from our laboratory demonstrated that under low affinity binding conditions (low Ca2+, low ionic strength, low pH), it is possible to detect multiple components in the dissociation curves, representing interconvertible states of the channel (20). For the current experiments, conditions (100 p~ Ca2+, 300 mM KCl, 20 mM MOPS (pH7.2)) were chosen to produce, at low occupancies, a single exponential component in the dissociation. It should be noted that the conditions that produce a single component in rabbit skeletal muscle produce multiple components in membranes derived from pig skeletal muscle, consistent with the higher apparent Kd for [3H]ryanodine binding to pig membranes compared to rabbit (20).

In experiments with three components (see below), fits of the data without constraining some of the parameters gave results that are difficult to interpret since there are then six variables and a number of equally good fits. Our approach to this problem was to determine rate constants for the fast and intermediate sites that fit all of the available data from similar experiments. These constants were then set, and data were fit allowing var- iation in the amplitudes of the different components and in the value for the slowest component. The latter was allowed to vary because it was easily determined in these experiments and because there could be a contribution of proteolysis over the extremely long times required to reach ti for this component.

At 130 nM [3Hlryanodine, there is primarily one exponential component to the dissociation curves and this has a dissociation rate constant (kl) of 0.0025 min-l (Fig. 4A). As occupancy increases (Fig. 4, B-D), two new components to the dissociation become apparent, a slow component ( k z = 0.00025 mix1) and a much faster one ( k 3 = 0.012 min-’1. The concentration de- pendence for the appearance of these two new components is very similar (Fig. 5, A and D). The results obtained with two different membrane preparations in independent experiments are shown in Fig. 5. If the bound [3H]ryanodine is diluted into a solution containing 100 p~ ryanodine, the intermediate component cannot be detected and there is much more of the slow component, suggesting an interconversion between these two components (Fig. 5, B and E ) . The relative amount of the fast component at each F3H1ryanodine concentration is, however, essentially the same as that detected by dilution alone (Fig. 4, E-H, and Fig. 5, B and E), suggesting that full occupancy of sites does not alter the rate of dissociation of [3H]ryanodine bound to this site. If negative cooperativity by binding of [3Hlryanodine at initially identical sites occurs, then, upon occupation of all four sites on the tetrameric receptor by ryanodine, the rate of dissociation from each of these sites should be identical. Scatchard analysis of the amount of the fast component bound at the different ligand concentrations (Fig. 5, C and F ) suggests that this component could give rise to the low affinity binding site detected in the Scatchard plot shown in Fig. 2.

Association of FHIRyanodine-If the binding of ryanodine at a low affinity site slows the dissociation of [3H]ryanodine bound at the high affinity site, then the only way the low affinity site could have arisen from one of four initially identical sites is if the binding at the high affinity site very greatly slowed the rate of association of ryanodine to the other site(s). If this does not occur and, if there are four initially identical sites, the dissociation data would have to be interpreted as indicating positive cooperativity. Is there any evidence for a slowing of the association rate with increasing occupancy? Association curves for the binding of L3H1ryanodine at concentrations that result in equilibrium occupancy values from 2.7 to 81.3% are shown in Fig. 6. The kobs plot is shown in Fig. 7. The kobs plot is linear, and the values obtained for K1 and k l from this curve can be used to calculate a Kd that is in good agreement with the Kd for

:m :m :Fi :Fi E. 130 nM. diluted into 1OOuM F. 230 nM, dilutcd into lOOuM G. 630 nM. diluted into lOOuM H. 1130 n ~ , diluted into

n o m

n o m

u v n o rn n o v m

C E C - -1 - -1 - -1

u C - -1

-1 -1 -1 -1

-3 0 M 0 l w o 1 . w two two Jwo an00

-3 o BOO low ~ M O awn 1.00 aooo JDOO o MOO loon (MOD m o o ~ M O soon JSOO -a 0 .Qo 1000 1.Qo *ow 1100 soon asw

-3

TIME (minutee) TIME (minutes) TIME (minutee) TIME (minutes)

FIG. 4. Dissociation of bound [SHlryanodine as a function of occupancy. SR membranes were incubated overnight (15 h) a t room temperature (23 "C) with L3H1ryanodine (130-1130 nM, each having the same amount of labeled ryanodine and an increasing concentration of unlabeled ryanodine) in a binding buffer containing 300 mM KCl, 100 p~ Ca2+, 50 mM MOPS (pH 7.2), 100 pg/ml BSA and protease inhibitors. At t = 0,50 pl of membranes were diluted into 80 ml of buffer with (E-H) and without (A-D) 100 p~ unlabeled ryanodine. Aliquots (1 ml) were filtered

membranes were incubated with 130 nM [3Hlryanodine. In B and F, SR membranes were incubated with 230 nM L3H1ryanodine. In C and G, SR at the indicated times. Filtration and scintillation counting were performed as described under "Experimental Procedures." In A and E , SR

membranes were incubated with 630 nM [3Hlryanodine. In D and H , SR membranes were incubated with 1130 nM c3H1ryanodine. Dissociation data are plotted as ln(B) uersus time, where B is in units of pmoYmg.

binding to the high affinity site obtained from equilibrium binding (Fig. 8).

At very high ligand concentrations, one would predict an instantaneous jump due to the binding to the high affinity site, followed by an extremely slow occupation of the low affinity site. Association at high [3H]ryanodine concentrations is shown in Fig. 9. A slow component is detected at ligand concentrations much higher than required for full occupancy of the high affinity site. The difficulty in determining the association rate constant for these slow reactions is that it is extremely difficult to reach a true equilibrium for binding. The value for the association rate constant for the slowly associating component cannot be determined. These experiments can only be used to demon- strate that there appears to be a slowly associating component, and we cannot, from these experiments, distinguish between two different but allosterically coupled sites and initially identical sites.

Low Affinity Binding to t-!hbuZes-If the low affinity site is distinct but allosterically or sterically coupled to the high affinity site, it is possible that it could either be on the channel itself or on a closely associated protein. The finding that the purified ryanodine receptor has low affinity binding sites ar- gues that the site is on the receptor itself. Analysis of [3Hlry- anodine binding to t-tubule membranes, however, demon- strates that low affinity binding sites for [3H]ryanodine exist in these membranes even though the high affinity sites for [3H]ryanodine are less than 2 pmol/mg (Fig. 10). The apparent Kd for the low affinity site is 2.3 * 1.8 p~ (n = 31, and the B,, in t-tubule membranes is 12.6 * 3.4 pmoVmg (n = 3). Other support for a ryanodine binding site in t-tubules comes from the finding that high concentrations of ruthenium red or ryano-

dine, but not neomycin, inhibit the binding of [3H]PN200-110 to t-tubule membranes (Fig. 11). The ICso for ruthenium red and ryanodine is 3.2 and 50.1 VM, respectively. These data suggest that low afiinity ryanodine binding sites may exist on proteins other than the Ca2+ release channel.

DISCUSSION

[3HlRyanodine binding can be used to monitor changes in the functional state of the Ca2+ release channel (7,9, 20). Evidence from several laboratories has suggested that there is only a single high affinity binding site for [3H]ryanodine per tetrameric channel in SR membranes (2, 7-16). We have shown that [3H]ryanodine binds with different affinities to the different functional states of the Ca2+ release channel (7, 20) and have proposed that, in any given incubation condition, the apparent K d for [3Hlryanodine binding to the Ca2+ release channel is a weighted average of the Kd values for the binding to each of interconvertible functional states of the protein. If nonspecific binding is defined in such a way as to exclude low affinity binding, a Scatchard plot of the binding to the high affinity site is always linear, and channel modulators alter the apparent Kd without any effects on &,,.

In the absence of AMP-PCP (or ATP) and/or at low ionic strength and low pH, the dissociation and association kinetics for [3H]ryanodine binding to SR membranes are characterized by multiple components and the relative ratios of these components can be manipulated by Ca2+ and other channel modulators (20). The ratio of the components of the dissociation kinetics relative to one another is not altered by the extent of occupancy of the high affinity site, again arguing for preexist- ing interconvertible states. Binding conditions can be chosen

Low Affinity Ryanodine Binding Sites

D

20979

A

3.0

2.5

E 2 2.0 - 0 E 1.5 U

0 1.0 z 3

0.5

0.0

B

l - 0

L [3H]-RYANODINE nM

3.0 - I I i

0 IIRIpxEDUTl rn lrm

';;; 2.5 0 SLOW

- 0

E 1.5 -

v

0 500 1000

[3H]-RYANODINE nM C.

n

E" 1 2 -

2 1 0 -

E 6 -

- a

0 z 3 4 - 0 m 2 -

v 6 -

01 0 500 1000

[3H]-RYANODINE nM E

[3H]-RYANODINE nN

F

0.014

0.012

0.002

W W E L

0.002

2 0.001 z 3 0

0.001

0.000

BOUND (pmole/mg) BOUND (pmole/mg) FIG. 5. The relative amounts of the three components of dissociation as a function of ryanodine concentration. Dissociation curves

from two representative experiments were fit with the sum of three descending exponentials. Panels A-C represent the experiment shown in Fig. 4, while LLF are data obtained with different membrane fraction which had a higher number of binding sites per milligram of protein. A and D show the amount of each component at the different ligand concentrations when dissociation is by dilution alone. B and E show the amounts of each component at the different ligand concentrations when the dissociation is into 100 PM ryanodine. C and F represent Scatchard plots of the amount of the fast component at the different ligand concentrations. 0, slow dissociation component; 0, intermediate component; W, fast component.


ASSOCIATION WJTH INCREASING CONCENTRATIONS OF [3H]-RYANODINE

14000 I I

12000

EO00

4000

2000

0 0 200 400 E O 0 800 1000

TIME (minutes) FIG. 6. Association of i3H1ryanodine as a function of occu-

pancy. SR membranes (50 pg) were added to 50 ml of 0.3 M KCl, 100 V M Ca2+, 50 mM MOPS (pH 7.2), 100 pg/ml BSA, and protease inhibitors containing 0.04 (01, 0.09 (01, 0.22 (V), 0.45 (VI, 0.89 (m), 3.0 (01, or 5.9 (A) nM L3H1ryanodine; 1-ml fractions were filtered at the indicated times. Filtration and scintillation counting were performed as described under “Experimental Procedures.”

0.01 5 I 1

0.010 -

n m

Y 0

0.000 , 0 1 2 3 4 5 8

[3H-RYANODINE] nM

were obtained from nonlinear curve fits of the data in Fig. 6 as described FIG. 7. kobs as a function of ryanodine concentration. kobs values

under “Experimental Procedures.”

such that the kinetics using low ligand concentrations produce primarily only a single component to both the association and dissociation curves. In membranes derived from rabbit skeletal muscle, these conditions are 300 mM KCl, 100 PM Ca”, 20 mM MOPS (pH 7.2).

High and low affinity [3Hlryanodine binding sites are important to the function of the Ca2+ release channel. Binding of ryanodine to the high affinity site locks the channel into an open, low conductance state ( 6 , 10, 12), whereas occupation of the low affinity site either blocks or closes the channel (10,111. Several reports have indicated that the ratio of high to low affinity sites is 1 to 3, supporting the idea that the three low affinity sites arise from the negatively cooperative binding of [3H]ryanodine at one of four initially identical sites. Quantitat- ing this ratio is difficult for four reasons. 1) The nonspecific binding is extremely high at the concentrations required to define the low affinity site. 2) It has not been possible to satu- rate the low affinity sites, since the solubility of ryanodine at these high concentrations is questionable. 3) The extremely slow association rate for the low affinity site makes it difficult

30 k i I

5 t \ i 0 1 \ I

0 10 20 30 40

BOUND (pmole/mg) FIG. 8. Scatchard analysis for association experiment. Equilib-

rium values for the binding in the experiment in Fig. 6 were used to calculate Scatchard values as described under “Experimental Proce- dures.” The calculated Kd is 1.1 nM, and the Bmax is 33.9 pmollmg.

4 1 I 1

v 200 nY 0 DO0 nY H 1.7 UY

n m W c

- 3 C’ I 0 200 400 E O 0 800 1000

TIME (minutes)

FIG. 9. Association at high [SHlryanodine concentrations. SR membranes (4.6 mg/ml) were incubated overnight (15 h) in 100 VM CaZ+, 1 mM AMP-PCP, 100 pg/ml BSA + protease inhibitors, 300 m~ KC1,50 m~ MOPS (pH 7.2) at 4 “C. At t = 0, 500 p1 of the membranes were added to 5 ml of the same buffer containing 28 nM [3H]ryanodine and increasing amounts of unlabeled ryanodine to produce the indicated concentration of ryanodine (V, 200 m; 0, 900 nM; B, 1.7 w). Aliquots (200 p1) were filtered at the indicated time. Filtration and scintillation counting were performed as described under “Experimental Proce- dures.”

to reach equilibrium before proteolysis becomes a problem. 4) Other low affinity binding sites appear to exist. Although our data are most consistent with one high affinity site for each low affinity site, we concede, because of the limitations of the experimental approach, that it is possible that there could be more than one low affinity site for each high affinity site.

The low affinity [3H]ryanodine binding site does not appear to be detectably altered by AMP-PCP, neomycin, Ca2+, or SO:- under conditions where these compounds dramatically affect the apparent affinity of the high affinity site for L3H1ry- anodine. This is in agreement with the findings of Emmick et al. (211, who showed that the effects of AMP-PCP on the acti- vating and inhibiting actions of ryanodine were consistent with independent binding sites on the Ca2+ release channel. Fur- thermore, 8-alanyl-ryanodine exhibited only activator activity.

Our data obtained from replacing C1- with SO:- in the [3H]ryanodine binding assay are consistent with the findings of Sukhareva and Coronado (22), who found that C1- is important


0.0 0 2 4 6 8 1012141818202224282830

BOUND (pmoles/mg) FIG. 10. Scatchard analysis of the binding of tSHIryanodine to

rabbit skeletal muscle t-tubule and SR membranes. Membranes (75 pg) were incubated with ["Hlryanodine diluted 1: lOO with unlabeled ryanodine over a concentration range of 7.8 nM to 5 p~ in a binding buffer consisting of 0.3 M KCl, 20 mM MOPS (pH 7.2), 100 p~ Ca2+, 100 pg/ml BSA, and protease inhibitors overnight (15 h) at room temperature (23 "C). Nonspecific binding was defined in the presence of 100 PM unlabeled ryanodine. Filtration and scintillation counting were performed as described under "Experimental Procedures." The Kd and Bmax values for these plots are as follows. For t-tubule (0): Kdl = 8 n ~ , BmaXcl, = 1.2 pmol/mg, Kd2 = 1.2 p ~ , and = 7.8 pmol/mg. For SR membranes (0): Kdl = 5.3 x", Bmaxcl), 11.8 pmol/mg, Kdz = 1.2 PM, and Bmax(2) = 13.6 pmol/mg.

120 I I I

100 -

80 -

80 -

40 - 0 v

20 -

neornycln

0 -7 -8 -5 -4 -3

log[ll

[8HlPN200-110 binding to t-tubule membranes. A total of 10 pg of FIG. 11. Effect of neomycin, ryanodine, and ruthenium red on

t-tubule membranes were incubated with 1 nM ["HlPN200-110 in 2 ml of 50 mM MOPS (pH 7.4) for 2 h at room temperature (23 "C) in the dark. Nonspecific binding was defined in the presence of 1 p~ nitrendipine. Filtration and scintillation counting were performed as described under "Experimental Procedures." V, neomycin; 0, ryanodine; 0, ruthenium red.

for Ca2+ release from SR vesicles. The replacement of C1- caused a decrease in the apparent affinity of the high affinity site for [3H]ryanodine. This may indicate either that C1- is important for L3H]ryanodine binding or that SO:- at this concentration is less effective or inhibitory. Similar to AMP-PCP and neomycin, the effect of SO:- is primarily on the high affinity binding site.

Low Ca2+ concentrations decrease the apparent K d for the high affinity site to a value similar to that of the low affinity site. In dissociation experiments, we have previously shown that lowering the Ca2+ concentration in the dilution solution enhances the rate of dissociation of [3H]ryanodine bound to the high affinity site (20). The presence of high concentrations of

Cytoplasm RYANODINE

Closed Open Open Closed

State: A B C D Lumen

FIG. 12. A model for the allosteric regulation of ryanodine binding at one high and one low affinity site. State A , a closed channel, would predominate a t low Ca2+ concentrations. This state binds [3H]ryanodine with low affinity. Upon binding Ca2+, the equilibrium shifts to favor state B. This is an open channel. The rate a t which ["Hlryanodine reaches its binding site is much faster for this state than for stated. This state of the receptor is also stabilized by activators such as ATP. When ryanodine binds to the open channel (state C), it dissociates with an intermediate rate. The liganded channel has a reduced conductance. At high concentrations of ryanodine, the ligand binds to a second site (state D ). Occupation of this site either closes or blocks the channel and greatly slows the dissociation from the high affinity site. Dissociation of bound [3Hlryanodine from the second site is fast.

ryanodine in low Ca2+ dilution buffers still slows the dissociation of [3H]ryanodine bound to the high affinity site (data not shown), suggesting that, although similar in apparent affinity, the two sites are distinguishable even in low Ca2+.

To address more quantitatively the question of whether the low affinity sites arise from the binding of ryanodine a t one of four initially identical sites, we have utilized a kinetic approach in which we have chosen conditions under which the kinetics for binding to the high affinity site a t low ligand concentrations is characterized by a single exponential component. With the high affinity site fully occupied, further increases in the [3Hlryanodine concentration of the incubation have three effects on the dissociation of the bound [3H]ryanodine. 1) At high ligand concentrations a fast component to the dissociation appears. 2) There is also an increase in an extremely slowly dis- sociating component. 3) The intermediate component, which is the major component a t low ligand concentrations, disappears. If the dissociation is performed in the presence of 100 p~ ryanodine, only the fast and slow components are seen and amount of the fast component is unchanged while the slow component has greatly increased. If 100 PM ryanodine causes all sites to be fully occupied, then initially identical sites would require that the dissociation from all sites be identical and there should be only a single component to the dissociation. This is not the case; therefore, the most likely explanation is that the high and low affinity sites are not initially identical. Our association data are consistent with this interpretation.

A simplistic model based on the data presented in this paper and on previous results and which places the low affinity sites on the Ca2+ release channel itself is shown in Fig. 12. Our previous data (7,201 suggest that: 1) [3H]ryanodine binds with low affinity to the closed channel, which predominates a t low Ca2+ concentrations and that 2) Ca2+ drives the channel to an open conformation (state B), a state stabilized by channel activators such as AMP-PCP and which binds [3H]ryanodine with high affinity. [3HlRyanodine bound to state C dissociates with an intermediate affinity. At high [3H]ryanodine concentrations, a second site is occupied (state D), and this occupation either causes the channel to close (as represented in the model) or blocks the channel (not depicted). The occupation of this low affinity site (from which ryanodine dissociates with a relatively fast rate) either traps [3H]ryanodine bound at the high affinity site or in some other way hinders its dissociation such that the dissociation from the high affinity site is extremely slow. If there is only a single high affinity binding site and if negatively cooperative binding at one of four initially identical sites does


not occur, then the high affinity site is probably located in a region contributed to by all four subunits. If the location of the binding site is in the channel itself, its presence may lock the channel in an open conformation but impede the flow of ions through the channel resulting in a open, low conductance channel.

As the ryanodine concentration increases, the alkaloid binds to site 2, which, if there is only one site per tetramer, may also be in a region shared by the subunits. A second possibility is that there is indeed a potential binding site for [3H]ryanodine on each subunit but that occupation of the first site sterically hinders the occupation of other sites. Although this mechanism could conceivably explain the slow association to the second site, it is difficult to understand how this type of mechanism could slow the dissociation from the high affinity site to the extent shown in Fig. 3, especially if association to this second site is also slow.

The concentrations of ryanodine required for inhibition of Ca2+ release (>low) also inhibit the binding of [3HlPN200-110 to the dihydropyridine-binding protein, and we have demonstrated that low affinity binding sites exist in membranes containing very few high affinity [3H]ryanodine binding sites. In addition, we have demonstrated that magnesium and ruthenium red, compounds that inhibit binding of ryanodine to low affinity sites in SR membranes,2 inhibit the binding of [3HlPN200-110 to t-tubule membranes. Neomycin does not inhibit the binding of ryanodine to low affinity sites in SR mem- branes2 and has no effect on the binding of [3H]PN200-110 to t-tubule membranes. Our data suggest that low affinity binding sites are not exclusive to the ryanodine receptor and may exist on proteins associated with or part of the dihydropyridine- binding protein. Whether these t-tubule sites are similar to those in the SR is not known. One possible explanation is that the low affinity binding site is on a protein which associates with both the dihydropyridine-binding protein and the Ca2+ release channel. Since low affinity binding can be found in a

J. P. Wang, D. H. Needleman, A. B. Seryshev, and S. L. Hamilton, unpublished data.

purified ryanodine receptor preparation, this protein would have to copurify with the ryanodine receptor. The model shown in Fig. 12 would then need to be modified to incorporate either a blocking or allosteric closing of the channel by ryanodine bound to an accessory protein, rather than to the channel itself. This possibility of a binding site on an associated protein is currently being investigated.

Acknowledgements-We thank Dr. William Schilling, Dr. Irena Sery- sheva, Dr. Steen Pedersen, Celetta Callaway, and Alexander Seryshev for many helpful discussions, Celletta Callaway for expert technical assistance, and Ginger May for help in preparation of the manuscript.

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[3h]ryanodine binding sites to high affinity sites on the skeletal

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