Brain Research, 561 (1991) 181-191 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117017U BRES 17017

181

Research Reports

Pharmacological characterization of the specific binding of [3H]ryanodine to rat brain microsomal membranes Ildiko Zimanyi* and Isaac N. Pessah Department of Veterinary Pharmacology and Toxicology, University of California, Davis, CA 95616 (U.S.A.) (Accepted 14 May 1991) Key words: Ryanodine; Inositol 1,4,5-trisphosphate; Brain microsome; Intracellular Ca 2+

High-affinity binding of [3H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein (M r -320 kDa), similar to that of ryanodine receptor from muscle sareoplasmic reticulum (SR). In the presence of high salt (1 M KCI), [ 3H]ryanodine binds to low density (0.8 M sucrose) cortical mierosomal fraction with high affinity (K a 1.5 nM), and with the highest capacity (Bronx330 fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca 2+ dependent (EDs0 1/zM) and inhibited by Mg2+ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of [ 3H]ryanodine. At low Ca 2+ concentration caffeine and daunorubicin enhance the binding of [3H]ryanodine. The inositol 1,4,5-trisphosphate (IP3) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of [ 3H]IP3, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatie reticulum (ER) Ca2+ release channel. INTRODUCTION Endoplasmic reticulum (ER) of a wide variety of cell types has the ability to sequester and mobilize significant amounts of intracellular calcium thereby mediating cell activation. Two types of ligand-gated calcium release channels are localized in regions of E R which mobilize the release of intracellular calcium pools; (1) those mediated by the second messenger inositol 1,4,5-trisphosphate (IP3-induced release) and (2) those mediated by calcium itself (Ca2÷-induced Ca 2÷ release; CICR). The IP 3 receptor is known to mediate calcium release from E R in a number of cell types found in the nervous system 35"39, in smooth muscle 6, liver 37'38 and adrenal chromaffin cells 3°'36. The IP 3 receptor, first purified by Supattapone et al. 39 from rat cerebellar cortex, has been recently reconstituted in lipid vesicles and shown to comprise a ligand-gated Ca 2+ channel 9. Furuichi et al. 12 have recently cloned the IP 3 receptor c D N A from mouse cerebellum and have expressed the 'P400' protein in a neuroblastoma-glioma hybrid cell line. C I C R channels localized at sarcoplasmic reticulum

(SR) terminal cisternae of striated muscle are characterized by their ability to bind the plant alkaloid ryanodine with nanomolar affinity 1°'31. The [3H]ryanodine-binding sites are conformationally sensitive to Ca 2÷ and indeed are modulated by the same relevant ligands which mediate C I C R suggesting that the ryanodine receptor and the C I C R channel are synonymous 15"18'19'32. Cloning of the ryanodine receptor from skeletal muscle 4° has revealed striking structural similarities with the brain IP 3 receptor suggesting that ligand-gated Ca 2+ channels of E R may represent a new genetic class of channels involved in mediating cell activation 13. Important questions remain concerning possible pharmacological overlaps between the IP 3 and ryanodine receptors and whether the two receptors can coexist in the same cell. For example, the release of intracellular Ca 2+ stored in SR of smooth muscle and E R of peripheral neurons and adrenal chromaffin cells can be triggered by either caffeine (a C I C R channel agonist) or by IP 3 (refs. 5,6,41). The IP 3 receptors in these cells appear quite similar with respect to their structural and functional properties whereas direct demonstration of the existence

* On leave from the Institute of Experimental Medicine of the Hungarian Academy of Sciences, H-1450, Budapest, P.O. Box 67, Hungary. Correspondence: I. Zimanyi, Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616, U.S.A.

182 o f [3H]ryanodine r e c e p t o r s is lacking. In a d d i t i o n the m e c h a n i s m r e s p o n s i b l e for IP3-induced c o n t r a c t u r e s in striated muscle and its r e l a t i o n s h i p to the r y a n o d i n e rec e p t o r c o m p l e x is u n c l e a r 11. T h e e x i s t e n c e o f b o t h I P 3 and r y a n o d i n e / C a 2+ sensitive r e c e p t o r s in the s a m e n e u r o n a l cells raises the q u e s t i o n w h e t h e r t h e s e r e c e p t o r s m e d i a t e the release o f C a 2+ f r o m the s a m e o r d i f f e r e n t pools, a n d w h a t the physiological role o f the r y a n o d i n e r e c e p t o r is in the central n e r v o u s system 29. S e v e r a l labo r a t o r i e s h a v e r e c e n t l y identified [ 3 H ] r y a n o d i n e - b i n d i n g sites h a v i n g n a n o m o l a r affinity in b r a i n m i c r o s o m e s 3'7"8' 20,24. T h e r e c e p t o r is i s o l a t e d in the 30S fraction by sucrose density g r a d i e n t c e n t r i f u g a t i o n and exhibits C a 2÷ c h a n n e l activity w h e n r e c o n s t i t u t e d in lipid p l a n a r bilayers 3"2°. M c P h e r s o n and C a m p b e l l 24 h a v e r e c e n t l y i m m u n o p r e c i p i t a t e d r y a n o d i n e r e c e p t o r s isolated f r o m rabbit b r a i n m i c r o s o m e s with s h e e p p o l y c l o n a l a n t i b o d i e s dir e c t e d against purified skeletal muscle r y a n o d i n e r e c e p tor. I m m u n o h i s t o c h e m i c a l localization o f I P 3 r e c e p t o r s in avian P u r k i n j e cells r e v e a l that t h e y are p r i m a r i l y localized in the r o u g h e n d o p l a s m i c r e t i c u l u m and in the d e n d r i t i c spines, w h e r e a s r y a n o d i n e r e c e p t o r s f o u n d in avian n e u r o n s are p r i m a r i l y localized to the intracellular m e m b r a n e s o f the p e r i k a r y o n , the d e n d r i t i c a r b o r and the a x o n 7,s. T h e p r e s e n t w o r k is u n d e r t a k e n to c h a r a c t e r i z e the distribution o f [3H]ryanodine- a n d [3H]IP3-binding sites in sucrose g r a d i e n t purified m i c r o s o m a l fractions f r o m 4 regions o f rat brain, and to c o m p a r e t h e i r p h a r m a c o l o g ical specificities. MATERIALS AND METHODS

layers. Each fraction was diluted with 5 mM HEPES buffer pH 7.4 to approximately 0.3 M sucrose concentration and was pelleted at 110,000 gmax for 60 min at 4 °C in a Beckman TI 50.2 rotor. The final pellets were resuspended in homogenization buffer at approximately 3-4 mg/ml protein concentration, 0.25 ml aliquots were rapidly frozen in liquid N 2 and stored at -80 °C until .used. Protein concentrations were determined by the modification of the method of Lowry et al. 22. Protein was precipitated by 4 vols. of 2% perchloric acid, pelleted at 92,000 g and resuspended in 1 N NaOH to the original volume prior the measurement.

Equilibrium binding of [3H]ryanodine Binding of [3H]ryanodine (spec. act. 60 Ci/mmol, New England Nuclear) was assayed as described by Ashley 3. Briefly, 100/~g of brain microsomal protein was incubated in 1 ml final volume of assay buffer consisting of 20 mM K-PIPES pH 7.4, 1 M KCI, 1 mM MgATP, 200/~M CaCl 2 (resulting in 100/~M free Ca z÷, calculated according to the SPECS computer program), 100/~M PMSF and 0.5-1 nM [3H]ryanodine unless indicated otherwise. Equilibrium binding constants were determined in two ways; (1) titration of 0.05-16 nM [3H]ryanodine of constant specific activity (hot titration curve), and (2) titration of 0.25 nM to 16/tM unlabeled ryanodine against the binding of 0.5 nM [3H]ryanodine (cold titration curve). Non-specific binding was determined in the presence of 100-fold excess of unlabelled ryanodine and averaged 20% of total binding. The incubation mixture was shaken for 2 h at 37 °C. The binding was terminated by rapid filtration using Brandel cell harvester through Whatman GF/B glass fiber filters. The filters were washed twice with 2.5 ml ice-cold wash buffer (20 mM Tris-HCl, 250 mM KC1, 15 mM NaCI, 50/~M CaCl 2 pH 7.1) and radioactivity was counted by liquid scintillation with an efficiency of 43%. Filter binding measured in the absence of protein was negligible.

Measurement of association~dissociation kinetics The rate of association of [3H]ryanodine binding was measured by quenching the reaction by rapid filtration at times ranging from 5 to 300 min after the addition of brain membranes. Dissociation of [3H]ryanodine from the receptor equilibrium complex was determined by equilibrating - 1 nM [3H]ryanodine with membranes for 2 h at 37 °C, followed by the addition of 1000-fold excess of unlabeled ryanodine to the incubation mixture or diluting the assay medium 100-fold and determining residual specific binding at subsequent times ranging from 5 min to 5 h.

Preparation of rat brain microsomes Rat brain microsomal membranes were prepared by modification of the method of Alderson and Volpe a. Sprague-Dawley rats (150200 g) were killed by decapitation, the brain was quickly removed and dissected free from meninges and 4 brain regions were sectioned: frontal cortex, hippocampus, brainstem and cerebellum. Each region was homogenized in 10-fold (w/v) of ice-cold 0.32 M sucrose, 5 mM HEPES buffer pH 7.4 (homogenization buffer) containing 100/~M phenylmethylsulfonylfluoride (PMSF) and 10/~g/ml leupeptin in a Potter-Elvehjem teflon-glass homogenizer. Homogenization was accomplished with 5 high-speed up and down strokes using an electric drill. The homogenate was spun at 1000 gmax for 10 min at 4 °C, the supernatant was collected and the pellet was resuspended in half the original volume of homogenization buffer using 2-3 strokes of the homogenizer, and repelleted. The first and second supernatants were combined and centrifuged at 17,000 gmax for 55 min in a Beckman TI 50.2 rotor at 4 °C, and the third supernatant was centrifuged at 100,000 gmax for 60 min at 4 °C using the same rotor. The pellet (P3) was resuspended in homogenization buffer and 30 mg protein of each brain region was layered on top of discontinuous sucrose gradients consisting of 5 ml 0.8 M, 6 ml of 0.9, 1.1 and 1.3 M and 5 ml of 1.6 M sucrose in 5 mM HEPES buffer pH 7.4 containing 10/~g/ml leupeptin. The gradients were spun in a Beckman SW-28 rotor at 20,000 rpm for 13 h. Three fractions were collected, fraction f-1 from the 0.8 M sucrose layer, fraction f-2 from the interface of 0.9 and 1.1 M sucrose layers and fraction f-3 from the interface of 1.1 and 1.3 M sucrose

Analysis of binding data Equilibrium binding data from saturation analysis were fitted to a one- or two-site model, and the dissociation constants (Kd), maximal binding capacities (Bmax), and Hill coefficients (nn) were determined by non-linear regression analysis using the LIGAND computer program. The first order association rate constants (k +a) were calculated as described by Bennett 4 based on the apparent association rate constant (kobs), determined by the ENZFITI'ER computer program, and the dissociation rate constants (k_0, which were calculated by least-squares linear regression analysis from In(SB/SBo = k_lt, where SB is specific binding at time t and SB o is specific binding at time 0. When dissociation proceeded in a biphasic manner, a second-order exponential was fitted with the ENZ F I I T E R computer program. At least 8 different concentrations of drugs were tested on equilibrium binding of [3H]ryanodine to brain microsomes. ICs0 and EDs0 values were computed using the ENZFITTER computer program.

Equilibrium binding of [3H]IP3 [3H]IP3 binding was measured on ice in a medium containing 50 mM Tris-HC1, pH 8.3, 1 mM EDTA and 100 mM KCI in a final volume of 0.5 ml as previously described 1. Total [3H]IP3 binding to 500/~g microsomal protein was measured in the presence of 50 nM [3H]IP3 (spec. act. 2500 dpm/pmol) only, non-specific binding was determined in the additional presence of 5/~M unlabeled IP 3. After 30 min incubation on ice the reaction mixture was rapidly ill-

183 tered through Whatman GF/B glass fiber filters using vacuum assisted Millipore single manifold filtration system and washed once with 2.5 ml ice-cold buffer. The bound radioactivity was measured by liquid scintillation spectrometry with an efficiency of 43%.

SDS-polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed in 3-17% linear gradient polyacrylamide gels with a 3% stacking gel, in a BioRad Mini-Protean Dual Slab Cell (Bio-Rad, Richmond, CA) with the buffer system of Laemmli a7. Gels were stained with Coomassie blue.

Spectrophotometric determination of

Ca 2+

TABLE I

Regional distribution of the binding of [3H]ryanodine and [3HJIP3 in rat brain Equilibrium binding of 0.25-40 nM [3H]ryanodine and binding of 50 nM [3H]IP3 was assayed as described in Materials and Methods. Data are the average of 3 independent experiments each performed in duplicate -+ S.E.M., where the S.E.M. is missing in that case the experiment was performed only once in duplicate. n.d., not determined.

Binding of [3H]ryanodine

release

Ca 2+ loading of brain microsomes and subsequent release of in-

travesicular Ca 2+ was measured by the method of Palade et al. 29. Rat cortical or cerebellar microsomes (100-150/,g of protein) were placed in a cuvette thermostated at 30 °C containing 970/A of 40 mM KCI, 8 mM K-MOPS, 62.5 KH2PO 4, 250/~M antipyrylazo III, 1 mM MgATP, 25/~g/ml creatine phosphokinase and 5 mM phosphocreatine at pH 7.0 (final volume 1 ml). The mixture was allowed to equilibrate for 1 min with constant stirring. Changes in the free Ca 2+ concentration were monitored by measuring the absorbance of antipyrylazo III at 710 nm and subtracting the absorbance at 790 nm at 1 s intervals using a diode array spectrophotometer (Model 8452A, Hewlett Packard, Palo Alto, CA). In some experiments, 3 reactions were monitored at 4.5 s intervals by utilizing the automated multi-cell transporter. The subtracted functional wavelengths, in absorbance units (AU), were displayed throughout the experiment in 'real time' on a monitor and concurrently stored by computer (Model 9000/300, Hewlett Packard) for subsequent analysis. The microsomes were actively filled by consecutive additions of 12.5 nmol CaC12 using a Hamilton syringe by allowing the absorbanee to return to the baseline between additions, or they were loaded with Ca 2+ in 3 ml batches at 30 °C for 2 h before use. In that case the loading time was exactly the same for each l ml aliquot used for spectrophotometric measurement. All the release experiments were performed at loading levels near the respective filling capacities of cortical (capacity = 0.3/~mol total Ca2+/mg protein) and cerebellar (capacity = 0.4 /~mol total Ca2+/mg protein) microsomes and kept constant within a dose-response titration. The total amount of Ca z+ loaded into the microsomes was quantified in two ways: first, by summing the downward deflections of absorbance in the loading phase of the experiment; second, by the addition of 2 /~g of the ionophore A23187 at the end of each experiment. Both methods were in excellent quantitative agreement (i.e. all of the Ca 2+ loaded into the vesicles during the loading phase was released by addition of ionophore). The release of intravesicular Ca 2+ was attempted with several concentrations of inositol 1,4,5-trisphosphate or ryanodine. The absorbance signals were calibrated by adding known amounts of CaC12 from a National Bureau of Standard stock to the complete transport mixture in the presence of 2 gg/ml A23187 to prevent Ca 2+ accumulation.

Cortex P3 f-1 f-2 f-3

Binding of [3HIIP3 (pmol/mg protein)

Ka (n M )

B,,m (fmollmg protein)

1.9 1.5 1.8 2.7

-+ 0.1 -+ 0.2 +-- 0.5 -+ 0.4

60 302 109 89

_+ 9 _+ 18 + 10 --- 13

0.4 0.3 0.3 --- 0.05 0.5 -+ 0.10

1.7 1.4 1.1 1.0

+- 0.1 _+ 0.2 -+ 0.1 + 0.2

58 122 85 60

--- 10 - 6 + 5 +_ 14

1.0 - 0.14 2.4 2.5 -+ 0.60 2.9 --- 0.01

Cerebellum P3

f-I f-2 f-3

Hippocampus f-1 2.1 -+ 0.3 f-2 n.d. f-3 n.d. Brainstem f-1 f-2 f-3

0.6 - 0.2 1.1 -+ 0.3 0.9 -+ 0.1

117 -+ 3 106 _+ 24* 75 -+ 16"

23 --_ 3 34 _+ 5 22 +- 5

1.1 0.4 0.2

0.2 0.4 -* 0.05 0.3 + 0.02

* Binding determined in the presence of 5 nM [3H]ryanodine 3 times in duplicate. RESULTS Specific high-affinity b i n d i n g

of [ a H ] r y a n o d i n e

has

b e e n e x a m i n e d in s u c r o s e - g r a d i e n t purified m i c r o s o m a l fractions o f 4 d i f f e r e n t rat b r a i n regions: c e r e b r a l c o r t e x , c e r e b e l l u m , h i p p o c a m p u s and b r a i n s t e m u n d e r o p t i m a l c o n d i t i o n s o f C a 2+ (100 /~M), M g A T P (1 m M ) , ionic s t r e n g t h (1 M K C I ) , p H (7.4) and P M S F ( 1 0 0 / z M ; a p r o t e a s e i n h i b i t o r ) which w e r e e x p e r i m e n t a l l y d e r i v e d (see b e l o w ) . T a b l e I s u m m a r i z e s e q u i l i b r i u m b i n d i n g

Measurement of ATPase activity ATPase activity was measured spectrophotometrically by enzymatically coupling ADP production to the oxidation of NADH with phosphoenolpyruvate, pyrnvate kinase, and lactate dehydrogenase 2, 42

Materials [3H]Ryanodine (spec. act. 60 Ci/mmol and >98 purity by HPLC) and o-[inositol-l-3H(N)]-l,4,5-trisphosphate were obtained from New England Nuclear (Wilmington, DE), uhlabeiled ryanodine was purified by HPLC (>99%) from a commercial (Penick, Tacoma, WA) mixture of ryanodine and dehydroryanodine. Inositol 1,4,5trisphosphate was from Calbiochem (San Diego, CA). All other chemicals were of the highest purity commercially available.

c o n s t a n t s o b t a i n e d for t h e 3 s u c r o s e - g r a d i e n t fractions ( i s o l a t e d as d e s c r i b e d in ' M a t e r i a l s and M e t h o d s ' ) f r o m e a c h b r a i n region. T h e b i n d i n g c a p a c i t y o f t h e c r u d e fractions (P3) is l o w e r t h a n the b i n d i n g c a p a c i t y o f t h e r e s p e c t i v e s u c r o s e - g r a d i e n t purified fractions (Table I). T h e b i n d i n g c a p a c i t y for [ a H ] r y a n o d i n e is e n r i c h e d 5-fold o v e r t h e c r u d e m i c r o s o m a l f r a c t i o n in t h e lightest cortical f r a c t i o n ( f - l ) . C o r t i c a l f-1 m e m b r a n e s are > 2 . 5 - f o l d e n r i c h e d c o m p a r e d to c e r e b e l l a r a n d h i p p o c a m p a l f-1 fractions. In g e n e r a l , the d e n s e r m e m b r a n e fractions f-2 and f-3 h a v e significantly l o w e r c a p a c i t y to b i n d r y a n o -

184

kDa 340 ,-.

1

2

3

4

a high-molecular weight protein of M r - 320 kDa, similar to the high-molecular weight spanning protein of the JSR which is the ryanodine receptor and a protein of M r 100 kDa, similar to the Ca 2+ pump protein (M r - 106 kDa) in the JSR. The high-molecular weight protein -possibly the ryanodine receptor in rat brain -- is consistent with the results of McPherson and Campbell 24 obtained with rabbit whole brain, and solubilized ryanodine receptor protein under similar conditions. The specific binding of [3H]ryanodine to f-1 cortical membranes is highly dependent on the ionic strength of the assay medium. Binding in the presence of optimal Ca 2÷ (see below) is negligible with NaCI or KCI, alone or in combination, up to 130 mM. Above 300 mM salt the specific binding increases with the salt concentration and it is 36% higher at 1 M KC1 than at 1 M NaCI (data not shown). All experiments which follow were therefore performed in the presence of 1 M KC1. The binding of 1 nM [3H]ryanodine to cortical f-1 membranes is linearly dependent on the protein concentration in the range of 25-500/~g protein (Fig. 2A) and has a p H optimum between 7.2 and 7.6 (Fig. 2B). The binding of [3H]ryanodine to its receptor in muscle is highly dependent on the free Ca 2+ in the assay medium 32. Ca 2+ in the presence of physiological (1 raM) Mg 2+ also activates the binding of [3H]ryanodine to cortical f-1 membranes with an EDso of 1.1 -+ 6-fold lower capacity to bind [3H]IP3 (Table I). Detailed characterization of the binding of [3n]ryanodine has been performed with the f-1 cortical fraction because of its high binding capacity. SDS-polyacrylamide gel electrophoresis of rat crude cortical microsomes (P3) and the f-1 cortical fraction are compared with rabbit skeletal muscle junctional SR (JSR) vesicles in Fig. 1. The brain microsomal fractions exhibit 103 5-

A

'5-

B

;0-

== o

g

.)5-

10

0 0

100

200 Protein

300 (/Jg)

400

560

6

7

pH

Fig. 2. Protein- (A) and pH-dependency (B) of the binding of [3H]ryanodine. Binding of 0.5 nM [3H]ryanodinc to various (A) or 100 #g (B) cortical sucrose-gradient purified microsomes was performed as described in the Materials and Methods.

185 (Fig. 4). Inhibition is especially dramatic with ATP. These effects are not the result of altered buffering of C a 2+ since the Mg-adenine nucleotide complexes were titrated. Cyclic AMP is only slightly inhibitory above 1 mM (Fig. 4). Equilibrium binding of 0.05 nM to 16 /~M [3H]ryanodine reveals binding sites having a single affinity for the radioligand which were fully saturated above 5 nM. Scatchard analysis of the binding of 0.05-16 n M [3H]ryanodine (spec. act. 60 Ci/mmol) results in a K d of 1.3 --+ 0.2 nM and a Bmax of 309 +- 19 frnol/mg of protein in the cortical f-1 fraction (Fig. 5, Table I). The Hill coefficient of the equilibrium binding is not different from o n e ( n H = 1.0) (Fig. 5B). The association of [3H]ryanodine (0.5 nM) is complete within 80 min and remains stable for up to 5 h (Fig. 6A). The observed rate constant of association (kob~) is 0.042 + 0.015 min -1. Dissociation of the [3H]ryanodine-receptor equilibrium complex has been examined in two ways.

A 160.

j,~,..-~t-I

lo~

/• .

./

/

S~.b

~

]

~

1

M)

Addition of 1000-fold unlabeled ryanodine (1 #M) results in biphasic dissociation curves best fit by a double exponential decay which yields two dissociation rate constants (Fig. 6B, Table II). The dissociation constants (Kds) can be calculated using the fast and slow rate constants and ko~ based on the relationships:

k+ 1

=

k°b" - k_t k_1 and Kd = [L] k+l

where k+l is the association rate constant, kobs is the observed association rate constant, k_ 1 is the dissociation rate constant, and K d is the dissociation constant. The result of these analyses are summarized in Table II. Dissociation of [aH]ryanodine by 100-fold dilution of the assay medium results in monophasic dissociation with a tv2 of 63 - 7 min (Fig. 6C) and yields a calculated Kd of 0.5 nM (Table II). The rat brain ryanodine receptor is sensitive to known modulators of the ryanodine receptor of muscle SR. Ruthenium red inhibits the binding of [3H]ryanodine with an ICs0 of 0.6 -+ 0.1 #M (Fig. 7A). Caffeine, known to stimulate the binding of [3H]ryanodine in muscle SR by increasing the apparent affinity of the Ca 2+ activator site for C a 2+ (ref. 32) also stimulates the binding of [3n]ryanodine to cortical membranes. In the presence of optimal (100 #M) free Ca 2+, caffeine has a negligible effect on ryanodine binding (data not shown), but at low free Ca 2+ concentration (100 nM) it enhances the binding with an approximate ECs0 concentration of 7-10 mM

1'o~

~

8o

~ 2o

.~

°

i°

---4

1o

ii1 2

5

, !

1~ [Adenine nucleotide] (mM)

0

]

lb

180"

~Mg24] (mM) Fig. 3. The effect of C a 2+ (A) a n d M g 2+ (B) on the binding of [3H]ryanodine. Binding of 1 nM [3H]ryanodine to 100/~g cortical sucrose-gradient purified microsomes was measured as described in Materials and Methods. Free Ca 2+ and Mg2+ concentrations were titrated with EGTA based on stability constants provided by the SPECS computer program developed by Fabiato. Data shown are average of two independent experiments performed in duplicate.

Fig. 4. Adenine nucleotides have a biphasic effect on the binding of [3H]ryanodine. The effect of ATP (o), AMP-PCP (e), and cAMP (A) was studied on the binding of 0.5 nM [3H]ryanodine to 100/~g rat cortical sucrose-gradient microsomal fraction. At each concentration of the adenine nucleotides the free Ca 2+ and Mg2+ concentrations were 100/~M and 1 mM, respectively, based on the affinity constants of the Ca2+- or Mg2+-adenine nucleotide complex provided by the SPECS computer program. The representative experiments shown were performed in duplicate and repeated once with similar results.

186 (Fig. 7B). Accurate m e a s u r e m e n t of the ECs0 of caffeine is hindered due to limited solubility above 100 m M concentration. D a u n o r u b i c i n , a potent antineoplastic agent which activates the binding of [aH]ryanodine by a mechanism similar to that described for caffeine 33 stimulates

2

o

150"

o ~,

1 00-

~

50-

tx

~ J ~

•

~ I

,

•

w

•

¢ A

the ryanodine binding in rat brain microsomes at low Ca 2+ concentrations (100 nM). The EDs0 concentration of daunorubicin is 81 -+ 3 # M (Fig. 7C). D a u n o r u b i c i n becomes inhibitory at high concentration, a p h e n o m e n o n also observed in muscle SR preparations (Pessah et al.,

tO

0r

unpublished data). Heparin (1-100/~M), a well-known inhibitor of the binding of [3H]IP3 has no effect on the binding of [aH]ry-

,

=

,

i

i

_e

0

1

2

3

4

b

0

1

2

3

4

5

'

I

J,

O"

I,

I

m

.,1oI 100

-1.51

0 ~ "~

. ~

_

O. 100.~ 1'0 1 ~5 Free (nM)

I

/

200

'

0 0 2 3 4 5

aO0 2b

%

2

Ln [Ryanodine] Fig. 5. Equilibrium binding of 0.05-16 nM [3H]ryanodine to rat cortical sucrose-gradient purified microsomes. Binding of [3H]ryanodine to 100 gg of the first cortical fraction was performed as described in Materials and Methods. A: the saturating specific binding, inset shows the Scatchard analysis of the binding, K d = 1.06 nM, Bm~ = 290.6 fmoi/mg of protein, B, bound (fmol/mg of protein), B/F, bound/free (fmol/mg of proteirdnM). B: the Hill plot of the specific binding, n n = 1.00. Data shown are from a representative experiment performed in duplicate which was repeated twice with similar results.

TIME

(hr)

Fig. 6. Kinetic analysisof the binding of [3H]ryanodineto rat cortical sucrose-gradient purified microsomes. A: association curve of the binding of [3H]ryanodineto the first cortical fraction. Data points are from two independent experiments each performed in duplicate. B: dissociation of [3H]ryanodinefrom the binding sites of the first cortical fraction induced by 1 #M unlabeled ryanodine. Double exponential decay fit was performed by the ENZFITI'ER program, dotted lines represent the linear components. Data points are the average of two independent experiments each performed in duplicate. C: dissociation of [3H]ryanodinefrom the binding sites of cortical microsomes was induced by 100-fold dilution of the assay medium. Single exponential decay fit was performed by the ENZFITI'ER program. Data points are the average of two independent experiments each performed in duplicate. Kinetic constants are summarized in Table II. SBo is specific binding at time 0, SB is specific binding at time t. At 0.5 nM [3H]ryanodineSB0 was 102.3 ± 24 fmol/mg of protein.

187 completely prevented the vesicles from active filling. I P 3 (1-10/zM) induces release of Ca 2+ in a concentrationdependent manner (Table III). Under these experimental conditions ryanodine (1 nM to 1 mM), caffeine (2-20 mM) and daunorubicin (200/tM) failed to induce the re-

TABLE II Kinetic constants o f the binding o f 1 n M [3H]ryanodine to sucrose-gradient purified rat cortical microsomes

Binding of [3H]ryanodine, its association to and dissociation from the receptor was measured as described in the legend of Fig. 6. The association constants were calculated as described in text. Mode o f dissociation

Association rate constant (k +z) (nM-Z.min -t)

1/aM ryanodine -slow phase 0.0280 -fast phase 0.0134 100-fold dilution 0.0220

Dissociation rate constant (k_z) (rain -z)

Ka calculated (nM)

0.0025 +_ 0.0008 0.0233 - 0.0026 0.0111 _+ 0.0010

0.09 1.7 0.5

anodine to the f-1 cerebellar fraction (data not shown), although heparin (100 /zM) inhibited the binding of [3H]IP3 by 80%. Conversely, 10 :tM Ruthenium red, 20 mM caffeine, or 1/~M ryanodine had no effect on the binding o f [3H]IP3 to the f-1 cerebellar fraction (data not shown). Brain microsomal preparations (P3 fractions) were used to measure the IP 3 induced release of extravesicular C a 2+ after active loading of the vesicles with incremental additions of 12.5 nmol CaC12, o r in batches. In general, crude microsomes take up C a 2+ approximately 3-fold faster than the respective sucrose-gradient fractions f r o m t h e c e r e b e l l u m . C o r t i c a l c r u d e m i c r o s o m e s t a k e up 1.3 t i m e s less C a 2+ t h a n c e r e b e l l a r c r u d e mi-

0

~uthenlumred] ;M)

, ll

5

110

B

i

c r o s o m e s ( d a t a n o t s h o w n ) . M e a s u r e m e n t o f A T P a s e activity in cortical c r u d e m i c r o s o m e s s h o w e d t h a t t h e m a o

j o r i t y o f the u p t a k e is n o n - m i t o c h o n d r i a l , b e c a u s e t h e

;

[Caffeine]

(mM)1~

' IO0

rate o f A T P a s e activity in t h e p r e s e n c e o f 1 m M s o d i u m azide was r e d u c e d by o n l y 26% f r o m 0 . 8 3 8 / t m o l P i / m g c

o f p r o t e i r d m i n to 0 . 6 1 6 / ~ m o l P i / m g o f p r o t e i n / m i n , respectively. I P 3 i n d u c e s r e l e a s e o f C a 2+ f r o m the actively filled vesicles in the a b s e n c e o f p y r o p h o s p h a t e . phosphate

(1 m M ) ,

60-

Pyro-

n o r m a l l y u s e d in m u s c l e studies, ~4~s

TABLE III IP~ induces release o f Ca 2+ from actively filled brain microsomes in a concentration-dependent manner

Rat brain cerebellar microsomes were actively loaded with CaCI 2 as described in Materials and Methods and the release of Ca 2+ was triggered by addition of IP 3. The total amount of release was calculated as a ratio of IP3-induced and IP 3 + 2/ag A23187 induced release of Ca 2+. IP s concentration (/aM)

Ca2+ release rate (AU/mg protein~rain)

% o f total Ca e+ release

1 5 10

0.110 _+ 0.07 0.447 0.724 _+ 0.17

9.03 14.13 12.47

1

• •

20-

c

~

"~' "

;

lb

[Daunorublcln~uM) ]

1do

lo o

Fig. 7. The effect of Ruthenium red (A), caffeine (B), and daunorubicin (C) on the binding of [3H]ryanodine. The binding of 1 nM [3H]ryanodine to sucrose-gradient purified cortical microsomes was assayed as described in Materials and Methods. Ca 2+ concentration in the presence of Ruthenium red (A) was 100/aM, in the presence of caffeine (B) and daunorubicin (C) was 100 nM. Data presented are the average of two independent experiments each performed in duplicate.

188 TABLE IV Ryanodine does not alter the ATPase activity of rat brain cortical microsomes

ATPase activity of 20 /~g crude cortical microsomes was measured as described in Materials and Methods. Ryanodine concentration (I~M)

ATPase rate (Izrn°l Pi ~rag protein/min)

-

0.838 --- 0.076 0.964 _+ 0.044 0.948 - 0.064

10 100

lease of C a 2+ o r modify the IP3-induced C a 2+ release. Even though IP 3 at micromolar concentrations causes rapid release of Ca 2+, only about 14% of the total accumulated Ca 2+ is released (Table III). Ryanodine has no effect on the ATPase activity of cortical microsomes up to 100 #M concentration (Table IV). DISCUSSION Mobilization of intracellular Ca 2÷ stores sequestered in ER is essential to elevating free cytoplasmic Ca 2÷ and initiating cellular activation. Stimuli responsible for triggering the release of E R Ca 2+ pools originate at the plasma membrane where external chemical signals mediate receptor activation and membrane depolarization. Two pathways of signal transduction have been described which lead to ER Ca 2+ release n. In a wide variety of cells, including neurons, IP 3 is generated by receptormediated activation of membrane-bound phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate to IP 3 and diacylglycerol. IP 3 released to the cytosol binds with high affinity to an E R Ca 2÷ channel and promotes release of Ca 2÷ sequestered in the lumen of ER. In cardiac muscle, Ca 2÷ entry through voltage-dependent Ca 2+ channels is the signal for activating the ryanodine receptor and triggering CICR from SR stores 26. Hence, Ca 2÷ itself is an essential second messenger in excitation-contraction coupling in cardiac muscle n. In contrast, release of SR Ca 2+ in skeletal muscle is mediated by charge movement across the transversetubule membrane where dihydropyridine-sensitive voltage-dependent Ca 2÷ channels may play the role of voltage sensor, thereby directly transducing electrical information to co-associated ryanodine receptors at the triad junction H. The primary amino acid sequences of the IP 3 receptor 12 and the ryanodine receptor 4° show some homology in the C terminal region of the final transmembrane domain and scattered homologies in the large N terminal region. Although the subunit mass of the two receptors (based on cDNA) are different, 313 vs 523 kDa for the IP 3 and ryanodine receptor, respec-

tively, both have a fourfold symmetry. There is pharmacological evidence for the presence of IP 3 sensitive and non-IP3-sensitive intracellular Ca 2+ pools in neuronal cells 5'21"23"41. Non-IP3-sensitive pools are found in the peripheral 21'27"41 and central 14'23'25 nervous system and are shown to be mobilized by caffeine and inhibited by ryanodine in a use-dependent manner 21'27'41. The caffeine-sensitive calcium stores have been observed in the cell soma, while IP3-sensitive stores seem to be distributed equally between the cell soma and processes 23'41. The present work pharmacologically characterizes the ryanodine receptor found in sucrose-gradient purified cerebral cortex membranes, which exhibit the highest binding capacity for ryanodine among the investigated brain areas in rat (Table I). This study allows comparison of the rat brain receptor with receptors found in skeletal and cardiac muscle recently completed in our lab. SDSpolyacrylamide gel electrophoresis shows the presence of the putative - 3 2 0 kDa molecular weight subunit, which is enriched in the sucrose-gradient purified cortical fraction (25/~g) compared to the crude microsomes (50/~g) (Fig. 1). This - 3 2 0 kDa protein has been identified as the ryanodine receptor in rabbit brain microsomes having a - 3 0 S sedimentation coefficient and showing crossreaction with antibodies directed against SR ryanodine receptor 7,8,24. Skeletal muscle ryanodine receptor is highly sensitive to Ca 2+ and has an apparent affinity (K~vca2+) of about 8 #M in the presence of 1 mM Mg 2÷ AMP-PCP (ref. 32). The binding of [3H]ryanodine to cortical membranes can be fully activated by 5/~M Ca 2÷ and has a KdJca2+ of 1 y M which is 8-fold lower than in skeletal SR (Fig. 3A). At optimal Ca 2÷ concentration, Mg 2+ inhibits the ryanodine binding both in muscle 43 and in rat brain (Fig. 3B), but the brain is 20-fold less sensitive to Mg 2÷. Both muscle and brain ryanodine binding is very sensitive to the ionic strength of the assay medium, K + above 500 mM is specially stimulatory giving the highest specific binding with cortical microsomes. Adenine nucleotides modulate the ryanodine receptor complex in skeletal muscle by an allosteric mechanism, activating the binding of [3H]ryanodine by increasing the binding capacity 32. With brain microsomes, ATP and AMP-PCP have a biphasic effect on ryanodine binding, activating the binding up to 1 mM and inhibiting at higher concentrations. In the absence of adenine nucleotides [3H]ryanodine binding is 40% below maximum activation (Fig. 4). These observations are consonant with those by Kawai et al. 16 in rabbit cerebral microsomes. Scatchard analysis of the equilibrium binding of [3H]ryanodine and Hill transformation of the binding curve suggested the presence of one binding site with a Ku of 1.5 nM and a Hill number of 1.00 (Fig. 5). Ki-

189 netic analysis of the association of ryanodine to the receptor and dissociation by 100-fold dilution of the assay medium (Fig. 6) also suggests the presence of one binding site, however, the K d calculated from the rate constants is about two-fold less than the Kd measured in equilibrium binding studies (Fig. 5 and Table II). Interestingly, dissociation by 1000-fold unlabeled ryanodine results in a biphasic dissociation curve, suggesting the presence of two binding sites (Fig. 6B). The fast component of the dissociation curve represents the component of binding estimated in equilibrium experiments (1.7 vs 1.5 nM, respectively), whereas the slow component suggests the presence of sites of higher affinity. The calculated Ko of this high-affinity site (Table II) is too low to detect in equilibrium binding studies using 60 Ci/ mmol specific activity. Equilibrium binding studies performed in the presence of 0.5 nM to 400 #M ryanodine do not suggest the existence of sites having affinities lower than 2 nM (data not shown). Kawai et al. 16 have shown multiple binding sites in rabbit cerebral microsomes with KdS of 30 and 400 nM. In those experiments the lowest concentration of [3H]ryanodine used was 10 nM which was too high to be able to demonstrate the presence of higher affinity sites. However, all these observations indicate the existence of multiple binding sites for ryanodine in the brain as in the skeletal or cardiac muscle, where we have demonstrated the existence of multiple binding sites for the alkaloid on the channel complex having nanomolar to micromolar affinities 34. Ruthenium red, a potent inhibitor of [3H]ryanodine binding to skeletal SR, is 30 times less potent in the brain (Fig. 7), whereas in low Ca 2÷ the receptor activators caffeine and daunorubicin have potencies similar to those found in muscle 33, suggesting a similar mechanism of activation. Regional studies of the binding of [3H]IP3 and [3n]ryanodine indicate that in rat brain the cerebral cortex has the highest binding capacity for [3H]ryanodine and it is poor in [3H]IP3 binding, and the cerebellum has the highest binding capacity for [3H]IP3 binding which is less rich in ryanodine binding capacity (Table I). These data are consistent with the finding of Ellisman et al. 7,8 in avian brain. Further pharmacological studies on the binding of [3H]ryanodine showed that heparin, a well known inhibitor of the binding of [3H]IP3, failed to affect the ryanodine binding, as well as ryanodine itself, and the ryanodine binding inhibitor, Ruthenium red, and the activators caffeine and daunorubicin have no effect on the binding of [3H]IP3.

As previously demonstrated 29, we were able to release Ca 2+ from actively filled brain microsomal vesicles with micromolar concentrations of IP 3. The fact that only about 14% of the total Ca 2÷ is releasable with IP 3 indicates the possible existence of another Ca 2+ pool (Table III). However, under the present experimental conditions we were not able to demonstrate ryanodine- or caffeine-induced Ca 2+ release, nor an effect of ryanodine on the total ATPase activity (Table IV). The inability of ryanodine to immediately release intravesicular Ca 2+ may suggest that the binding protein does not form an ion channel in the brain, but findings by others contradict this speculation. Single channel records obtained with bovine 2° or rat (ref. 3, McPherson et al., personal communication) whole brain microsomal membranes fused in lipid planar bilayers provide evidence for the existence of Ca 2+ channels which are sensitive to both ryanodine and IP 3 indicating that the ryanodine-binding protein forms an ion channel and this is identical to the IP3-sensitive channel. The results presented here show qualitative similarities and quantitative differences of the [3H]ryanodine binding compared to that of striated muscle. These and the fact that ryanodine does not induce Ca 2÷ release from brain microsomes under similar experimental circumstances as it does from muscle SR vesicles are in agreement with possibly different functional properties of the ryanodine binding protein in the central nervous system. Pharmacological comparison with the binding of [3H]IP3 to the same microsomal membranes show no pharmacological cross-reactivity between the two binding sites. This along with the findings of others 7'8'14'24'25' 28.29 suggest that the two binding proteins are not identical. One possibility is that they represent two distinct Ca 2÷ pools, one the IP3-sensitive pool, the other the Ca2+-activated, caffeine- and ryanodine-sensitive pool which can act as a cytosolic buffering system for calcium loads 23. The existence of two distinct channels mediating Ca 2+ release from the endoplasmic reticulum is supported by the binding data presented here. However, the question remains as to the physiological role of the ryanodine binding protein in the CNS, and whether it is coupled directly to a Ca 2+ release channel.

ABBREVIATIONS

AU Bmax CICR

AMP-PCP fl, z-methyleneadenosine 5"-triphosphate

Acknowledgements. The authors would like to thank Kristy J. Gayman for her dedicated technical assistance. This work was supported in part by NIH Grant ES05002 and Biomedical Research Support Grant 90-19 to I.N.P.

absorbance units maximal binding capacity calcium-induced calcium release

190 CNS ECso EDTA EGTA HEPES IC50 IP 3 JSR K-MOPS

central nervous system concentration of drug causing 50% of the maximal effect ethylenediaminetetraacetic acid [ethylene-bis-(oxyethylenenitrilo)]tetraacetic acid N-2-hydroxyethylpiperazine-N "-2-ethanesulfonie acid concentration of drug causing 50% inhibition of the maximal binding inositol 1,4,5-trisphosphate junctional sarcoplasmic reticulum potassium 3-(N-morpholino)propanesulfonic acid

REFERENCES 1 Alderson, B.H. and Volpe, P., Distribution of endoplasmic reticulum and caiciosome markers in membrane fractions isolated from different regions of the canine brain, Arch. Biochem. Biophys., 272 (1989) 162-174. 2 Anderson, K.W. and Murphy, A.J., Alteration of the structure of the ribose moiety of ATP reduced its effectiveness as a substrate for the sarcoplasmic reticulum ATPase, J. Biol. Chem., 258 (1983) 14276-14278. 3 Ashley, R.H., Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers, J. Membr. Biol., 111 (1989) 179-189. 4 Bennett Jr., J.P., Methods in binding studies. In H.I. Yamamura, S.J. Enna and M.J. Kuhar (Eds.), Neurotransmitter Receptor Binding, Raven Press, New York, 1978, pp. 57-90. 5 Burgoyne, R.D., Cheek, T.R., Morgan, A., O'Sullivan, A.J., Moreton, R.B., Berridge, M.J., Mata, A.M., Colyer, J., Lee, A.G. and East, J.M., Distribution of two distinct Ca2+-ATPaselike proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells, Nature, 342 (1989) 7274. 6 Chadwick, C.C., Saito, A. and Fleischer, S., Isolation and characterization of the inositol trisphosphate receptor from smooth muscle, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 2132-2136. 7 Ellisman, M.H., Deerinck, T.J., Ouyang, Y., Beck, C.E, Tanksicy, S.J., Walton, ED., Airey, J.A. and Sutko, J.L., Identification and localization of ryanodine binding proteins in the avian central nervous system, Neuron, 5 (1990) 135-146. 8 Ellisman, M.H., Deerinek, T.J., Ouyang, Y., Beck, C.E, Tanksley, S.J., Walton, ED., Airey, J.A. and Sutko, J.L., Identification of ryanodine binding proteins in the avian nervous system: localization in Purkinje cells differs from IP 3 receptors, Soc. Neurosci. Abstr., 16 (1990) 1175. 9 Ferris, C.D., Huganir, R.L. and Snyder, S.H., Calcium flux mediated by purified inositol 1,4,5-trisphosphate receptor in reconstituted lipid vesicles is aliosterically regulated by adenine nucleotides, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 21472151. 10 Fleischer, S., Ogunbunmi, E.M., Dixon, M.C. and Fleer, A.M., Localization of Ca 2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 72567259. 11 Fleischer, S. and Inui, M., Biochemistry and biophysics of excitation-contraction coupling, Annu. Rev. Biophys. Chem., 18 (1989) 333-364. 12 Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K., Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P4oo, Nature, 342 (1989) 32-38. 13 Gill, D.L., Receptor kinships revealed, Nature, 342 (1989) 1618. 14 Glaum, S.R., Scholz, W.K. and Miller, R.J., Acute- and longterm glutamate-mediated regulation of [Ca + +]i in rat hippocampal pyramidal neurons in vitro, J. Pharmacol. Exp. Ther., 253

k+l k_t kobs nH K-PIPES PMSF SDS SR tl,,2 Tris

true association rate constant dissociation rate constant equilibrium dissociation constant observed association rate constant Hill coefficient potassium piperazine-N,N'-bis[2-ethanesulfonic acid] phenylmethyisulfonyl fluoride sodium dodecyl sulfate sarcoplasmic reticulum haiftime of dissociation Tris[hydroxymethyl]aminomethane

(1990) 1293-1302. 15 Inui, M., Saito, A. and Fleischer, S., Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures, J. Biol. Chem., 262 (1987) 1563715642. 16 Kawai, T., Ishii, Y., Imaizumi, Y. and Watanabe, M., Characteristics of [3H]ryanodine binding to the rabbit cerebral microsomes, Brain Research, 540 (1991) 331-334. 17 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685. 18 Lai, F.A., Erickson, H.P., Liu, Q.-Y. and Meissner, G., Purification and reconstitution of the calcium release channel from skeletal muscle, Nature, 331 (1988) 315-319. 19 Lai, F.A., Anderson, K., Rousseau, E., Liu, Q.-Y. and Meissner, G., Evidence for a Ca 2+ release channel within the ryanodine receptor complex from cardiac sarcoplasmic reticulum, Biochem. Biophys. Res. Commun., 151 (1988) 441-449. 20 Lai, F.A., Xu, L. and Meissner, G., Identification of a ryanodine receptor in rat and bovine brain, Biophys. J., 57 (1990) 529a. 21 Lipscombe, D., Madison, D.V., Poenie, M., Renter, H., Tsien, R.W. and Tsien, R.Y., Imaging of cytosolic Ca e+ transients arising from Ca 2+ stores and Ca 2÷ channels in sympathetic neurons, Neuron, 1 (1990) 355-365. 22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin. phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 23 Martinez-Serrano, A. and Satrustegui, J., Caffeine-sensitive calcium stores in presynaptic nerve endings: a physiological role?, Biochem. Biophys. Res. Commun., 161 (1989) 965-971. 24 McPherson, P.S. and Campbell, K.P., Solubilization and biochemical characterization of the high-affinity [3H]ryanodine receptor from rabbit brain membranes, J. Biol. Chem., 265 (1990) 18454-18460. 25 Murphy, S.N. and Miller, R.J., Two distinct quisqualate receptors regulate Ca 2+ homeostasis in hippocampal neurons in vitro, Mol. Pharmacol., 35 (1989) 671-680. 26 Nabauer, M., Callewaert, G., Cleeman, L. and Morad, M., Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes, Science, 244 (1989) 800-803. 27 Nishimura, M., Tsubaki, K., Yagasaki, O. and Ito, K., Ryanodine facilitates calcium-dependent release of transmiqers at mouse neuromuscular junctions, Br. J. Pharmacol., 100 (1990) 114-118. 28 Padua, R.A., Geiger, J.D., Wan, W. and Nagy, J.I., Pharmacological characterization of high affinity [ 3H]ryanodine binding sites to subcellular fractions of rat brain, Soc. Neurosci. Abstr., 16 (1990) 1175. 29 Palade, P., Dettbam, C., Alderson, B. and Volpe, P., Pharmacologic differentiation between inositoi-l,4,5-trisphosphate-induced Ca 2+ release and Ca z+- or caffeine-induced release from intracellular membrane systems, Mol. Pharmacol., 36 (1989) 673-680. 30 Palmer, S. and Wakelam, M.J.O., The Ins(1,4,5)P 3 binding site of bovine adrenocortical microsomes: function and regulation, Biochem. J., 260 (1989) 593-596.

191 31 Pessah, I.N., Waterhouse, A.L. and Casida, J.E., The calciumryanodine receptor complex of skeletal and cardiac muscle, Biochem. Biophys. Res. Commun., 128 (1985) 449-456. 32 Pessah, I-.N., Stambuk, R.A. and Casida, J.E., Ca2+-activated ryanodine binding: mechanism of sensitivity and intensity modulation by Mg2+, caffeine, and adenine nueleotides, Mol. Pharmacol., 31 (1987) 232-238. 33 Pessah, I.N., Dude, E.L., Schiedt, M.J. and Zimanyi, I., Anthraquinone-sensitized Ca 2+ release channel from rat cardiac sareoplasmic reticulum: possible receptor-mediated mechanism of doxorubicin cardiomyopathy, Mol. Pharmacol., 37 (1990) 503-514. 34 Pessah, I.N. and Zimanyi, I., Characterization of multiple [3H]ryanodine binding sites on the Ca 2÷ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action, Mol. Pharmacol., 39 (1991) 679-689. 35 Ross, C.A., Meldolesi, J., Milner, T.A., Satoh, T., Supattapone, S. and Snyder, S.H., Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons, Nature, 339 (1989) 468-470. 36 Rossier, M.E, Capponi, A.M. and Vallotton, M.B., The inositol 1,4,5-trisphosphate-binding site in adrenal cortical cells is distinct from the endoplasmie reticulum, J. Biol. Chem., 264 (1989) 14078-14084. 37 Spat, A., Bradford, P.G., McKinney, J.S., Rubin, R.P. and

Putney Jr., J:W., A saturable receptor for 32-p-inositol-l,4,5trisphosphate in hepatocytes and neutrophils, Nature, 319 (1986) 514-516. 38 Spat, A., Fabiato, A. and Rubin, R.P., Binding of inositol trisphosphate by a liver microsomal fraction, Biochem. J., 233 (1986) 929-932. 39 Supattapone, S., Worley, P.E, Baraban, J.M. and Snyder, S.H., Solubilization, purification, and characterization of an inositol trisphosphate receptor, J. Biol. Chem., 263 (1988) 1530-1534. 40 Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T. and Numa, S., Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor, Nature, 339 (1989) 439--445. 41 Thayer, S.A., Hirning, L.D. and Miller, R.J., The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro, Mol. Pharmacol., 34 (1988) 664-673. 42 Warren, G.B., Toon, P.A., Birdsall, N.J.M., Lee, A.G. and Metealfe, J.C., Reconstitution of a calcium pump using defined membrane components, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 622-626. 43 Zimanyi, I. and Pessah, I.N., Comparison of [3H]ryanodine receptors and C a 2+ release from rat cardiac and rabbit skeletal sarcoplasmic reticulum, J. Pharmacol. Exp. Ther., 256 (1991) 938-946.