Steven D. Kahl$, Terence Lewisll, Philip Bentleyll,. Michael ... correspondence should be addressed Howard Hughes Medical Inst., ..... K. Willard, H. E , Khanna.
Communication
THEJOURNAL OF BIOLCGICAL CHEMISTRY Vol. 269, No. 18, Issue of May 6, pp. 13076-13079, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Ca2+ concentration, which controls contraction. Ryanodine rePhotoaffinity Labeling of the ceptors have also been characterized in neuronal tissue, where Ryanodine Receptor/Ca2+ Releasethey are also thought to performan important role in regulating Ca2+ release from caffeine-sensitive Ca2+ stores. With the Channel with an Azido use of L3H]ryanodine binding assays, all three Ca2+release Derivative of Ryanodine* channels have been purified and shown to be very large homotetramers witha monomer subunit molecular massof -565 kDa (1-6). Upon reconstitution in planar lipid bilayers, these receptors form large conductance cation channels sensitive to nanomolar concentrations of ryanodine. The primary strucDerrick R. WitcherS, Peter S. McPhersonsx Steven D. Kahl$, Terence Lewisll, Philip Bentleyll, t u r e s of several ryanodine receptors have been determined Michael J. Mullinnix$,John D. Windassll, and from a number of species and tissues (7-12), a n d the expression Kevin P. Campbell$ ** of ryanodinereceptor cDNA in Chinesehamsterovary or From the +Howard Hughes Medical Institute, COS-1 cells has generated high molecular mass receptors for Department of Physiology a n d Biophysics a n d the [3Hlryanodine (13, 14). Recently, a sulfhydryl-gated 106-kDa $Program in Neuroscience, University of Iowa College of protein has been identified in the sarcoplasmic reticulum of Medicine, Iowa City, Iowa 52242 a n d IEENECA and reconstitution experiskeletal muscle (15). Purification Agrochemicals, Jealott's HillResearch Station, ments in planar lipid bilayers suggest that this 106-kDa proBracknell, Berkshire RG12 6 E x Great Britain tein may alsobe a Ca2+ release channel sensitive to ryanodine Ryanodine receptors/Ca2+ release channels play an (16). important role in regulating the intracellular free calciumBiochemically, ryanodinereceptors/Ca2+releasechannels Ry- have been shown tobe modulated by a number of compounds concentrations in both muscle and nonmuscle cells. anodine, a neutral plant alkaloid, specifically binds to including ryanodine (17-21). Other agents such as Ca2+,ATP, and modulates these Ca2+ release channels. In the work of a trit- M e , ruthenium red, caffeine, and calmodulin, for example, described here, we characterize the interaction of ryanodine ('H- have been demonstrated to directly regulate the ryanodine reium-labeled, photoactivable derivative labeled 10-0-[3-(4-azidobenzamido)propionyl]ryanodine ceptor (22-26). The determination of the primary structure of (['HIABRy)) withthe ryanodine receptor of skeletal, car- the ryanodine receptor has led to further biochemical characsites on the skeldiac, and brain membranes. Scatchard analysis demon-terizations of both the ATP- and Ca2+-binding is known about strates that this ligand binds to a single class of high etal ryanodine receptor (27,28). However, little the location of the ryanodine-binding site(s). affinity sites in skeletalmuscletriads.Furthermore, Photoaffinity labelingis a powerful tool for studying ligandcompetition binding assaysof ['Hlryanodine with skelTo obtain information about the etal, cardiac, and brain membranes in the presence of binding sites on receptors. increasing concentrationsof unlabeled ABRy illustrate binding site of ryanodine on the calcium release channel, we that this azido derivative of ryanodine is able to specifi- have produced a novel tritium-labeled azido derivative of rycallydisplace[SH]ryanodinefrom its binding site(& anodine that binds the ryanodine receptor with high affinity ATP, and KC1 on ['HIABRy and is localized to Analysis of the effectsof Ca2+, a 76-kDa COOH-terminal tryptic fragment of binding in triad membranes shows a similar modulation the receptor. of binding to that seen in these membranes with ['Hlryanodine. Photoaffinity labelingof triads with['HIABRy EXPERIMENTALPROCEDURES of resultedinspecificandcovalentincorporation Membrane Preparation-Triads from rabbit skeletal muscle, canine ['HIABRy into a 666-kDa protein that was shown to be cardiac sarcoplasmic reticulum vesicles, and crude rabbit brain memthe skeletal muscle ryanodine receptor. Digestion of the branes were purified as previously described (29, 30, 31). labeled ryanodine receptor revealed a ['HIABRy-labeledMaterials-The azido derivative of ryanodine, 10-0-[3-(4-azidobenz76-kDa tryptic fragment that was identified with anan amido)propionyl]ryanodine (ABRy),' was prepared as previously detibody directed against the COOH-terminal of the recep- scribed (32). A tritiated analogue C3H1AElRy)was made by Amersham tor. These results demonstrate that the 76-kDaCOOH- International by the use of tritiated 4-azidobenzoic acid at the last synthetic step. The specific activity of t3H1ABRy was 45 Ci/mmol, and terminaltrypticfragmentcontainsthehighaffinity the radiochemical purity was >93% as determined by thin-layer chrobinding site for ryanodine. matography on silica gel and reverse-phase chromatography. f3H1Ry(Received for publication, February 1, 1994, and in revised form, February 28, 1994)
anodine was from DuPont NEN. Horseradish peroxidase-conjugated secondary antibodies werefrom Boehringer Mannheim. All other The ryanodine receptor/Ca2+ release channel has been exten- chemicals were reagent-grade. PHJABRy Binding Assay-Triad membranes (50 pg) were incubated sively studied in both skeletal and cardiac muscles, where it plays a central role in the regulation of the intracellular free with 0.5-50 n~ t3H]ABRy for 1h at 37 "C in the presence or absence of 10 p~ ABRy in 250 pl of 10 mM sodium HEPES, pH 7.4, containing 0.5 * The costs of publication of this article were defrayed in part by the M KCl, 10 m~ ATP, and 0.8 p CaCl, (50 p free Ca''). Proteins were payment of page charges. This article must therefore be hereby marked then collected onWhatman GF/B filters using a Brandel Cell Harvester. "advertisement" in accordance with 18 U.S.C. Section 1734 solely to For specific experiments, the concentrations ofATP, Ca2+,and KC1 were indicate this fact. varied individually while keeping all other variables constant. The con7 Present address: Dept. of Cell Biology,Yale University School of centration of free divalent cations was determined using the method of Medicine, New Haven, CT 06536. Fabiato (33). ** Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed Howard Hughes Medical Inst., The abbreviations used are: ABRy, lO-0-[3-(4-azidobenzamido)proUniversity of Iowa Collegeof Medicine, 400 Eckstein Medical Research pionyllryanodine; PAGE, polyacrylamide gel electrophoresis. Bldg., Iowa City, IA 52242. Tel.: 319-335-7867;Fax: 319-335-6957.
13076
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Photoaffinity Labeling of the Ryanodine Receptor SDS-PAGE and Immunoblot Analysis-Proteins were analyzed by SDS-PAGE (3-12% gradient gels) using the buffer system of Laemmli (34). Gels were stained with Coomassie Blue or were transferred to nitrocellulose forimmunoblot analysis as previouslydescribed (35). Specificpolyclonal antibodies against the COOH-terminal 15-amino acid peptide of the skeletal ryanodine receptor (Rabbit 46) (5) were affinity-purified from Immobilon-Ptransfer strips of the cardiac ryanodine receptor or the COOH-terminall5-amino acid peptide conjugated to bovineserum albumin. Both of these affinity-purified antibodies give the same resultson immunoblots. Gelsused for fluorography were first stained with Coomassie Blue, destained, soaked in Enlightning (DuPont NEN), and dried under vacuum. The dried gels werethen exposed to Kodak XAR-5 film with an intensifjmg screen at -80 "C. Covalent Coupling of PHIABRy-Skeletal muscle triads (250 pg) were incubated in the dark with 15 m r3HIABRyfor 1h at 37 "C in the presence or absence of 10 w ryanodine in 1 ml of 10 nm sodium HEPES, pH 7.4, containing 0.5 M KCl, 10 -ATP, and 0.8 nm CaCl, (50 p free Ca"). The membranes were then pelleted at 400,000 x g for 15 min and resuspended in 1 ml of binding buffer with 5 m~ glutathione. The samples were placed in a plastic Petri dish at 4 "C and exposed to U V light (365 n m ) a t a distance of 1cm for45 min. The membranes were then centrifuged in a microcentrifuge a t maximum speed for 15 min at 4 "C. The pellets were resuspended in Laemmli sample buffer (34), and the proteins were separated by SDS-PAGE. Following electrophoresis, the gels were stained with Coomassie Blue and wereprocessedfor fluorography. Proteolytic DigestionSkeletal muscle triads (250 pg) were prelabeled with r3H1ABRy as described above. Membranes were then resuspended (2 mg/ml) in 50 II~Mammonium bicarbonate and incubated with trypsin (1:lOOOenzymdmembranes (w/w)) for1, 10, and 15 min at room temperature. In control experiments, trypsin was omitted. Reactions were terminated by the addition of Laemmli sample buffer (34), followed by boiling for 2 min. RESULTS AND DISCUSSION
The tritium-labeled, photoactivable ryanodine analogue ([3HlABRy)used in this work is shown in Fig. lA. This compound has the same structure as ryanodine, except for the addition of a n azidobenzamidopropionyl group at position 10. The azido group (Fig. lA, N3)provides it with thecapability to be covalently cross-linked to proteins. ['HIABRy also contains two tritium atoms (2') on the azidobenzene ring at positions 3 and 5, which allows detection of the specific binding of this compound to various membranes. To characterizethebinding of [3HlABRy to theskeletal muscle ryanodine receptor, Scatchard analysis was performed on skeletal muscle triads. ['HIABRy (0.5-50m)bound to skeletal muscle triads ina saturable manner(Fig. lB 1. Nonspecific binding was determined in the presence of 10 p unlabeled ABRy The values for Kd and B,, for triads (determined from three separate experiments) were 5.5 nM and 12.7 pmol/mg, respectively. These data demonstrate that l3H1ABRybinds with high affinity to a single class of receptors in triad membranes. These Kd and B,, values for L3H]Al3Ry are very similar to the values obtained for [3H]ryanodine binding in skeletal muscle triads (1). At least three differentryanodinereceptor genes are expressed in skeletal, cardiac, and brain tissues (Refs. 7, 9,and 11;reviewed in Ref. 36). Ryanodine is known to specially bind with high affinity to the skeletal and cardiac Ca2+release channels. This compound also binds with high affinity to themajor brain form of the ryanodine receptor, which is the cardiacisoform (26,311. To determine if ['HIABRy bound to the same site on the Ca2+ release channel in skeletal, cardiac, and brain membranes as ryanodine,competition bindingexperiments were performed with ['Hlryanodine in thepresence of increasing concentrationsof ABRy. Fig. 2 shows that unlabeled ABRy specifically inhibited [3Hlryanodine binding to triads, with a half-maximal inhibition at -37 nM. The half-maximal inhibition concentrations for ['Hlryanodine binding to cardiac and brain membranes were 89 and 25 m, respectively (Fig. 2).
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FIG.1. Structural comparison between ryanodine and azido derivative of ryanodine(ABRy)andScatchardanalysis of ['HIABRy binding to skeletal muscle triads. A, the structure of ryanodine (left) is compared with that ofABRy (right). The locationsof the tritium atoms on l3HIABRyare designated as T in the structureof ABRy. E , triad membranes (50 pg) were incubated with various concentrations ofr3H1ABRy (0.5-50 m) for 1 h at 37 "C as described under "Experimental Procedures."Specific binding was determined in the presence of 10 w unlabeled ABRy. All experiments were performed in triplicate, and a representative example is shown. Scatchard analysis from three separate experiments yielded apparent Kd and B,, values for triads of 5.5 m and 12.7 pmol/mg, respectively.BIF, bounufree. 100 0
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FIG.2. Competition of [SH]ryanodinebinding to skeletal, cardiac, and brain membranes with unlabeled ABRy. Skeletal muscle triads (10 pg; A), total cardiac microsomes (200 pg; W), or crude brain microsomes (500 pg; 0 )were incubated with increasing concentrations of unlabeled ABRy in thepresence of 1m F3H1ryanodine for1h at 37 "C as described under "Experimental Procedures." 100%binding is defined by the amount of [3Hlryanodinespecifically boundin the presence of the lowest concentration of unlabeled ABRy.All experiments were performed in triplicate, and a representative example of three separate experiments is shown.
These values are in the same range as the half-maximal inhibition concentration for ['Hlryanodine binding to triads by ryanodins (-20.0 m), demonstrating that ABRy and ryanodine bind to the same siteson each receptor. A number of compounds have beenshown to modulate ['Hlryanodine binding to theCa2+release channel. Increasing concentrations of both KC1 and ATP dramatically stimulate l3H1ryanodine binding to all forms of the ryanodine receptor
13078 A
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RG.3.Analysis of KCl,ATP, and Ca% effects on [%lABRy binding to skeletal muscle membranes. [3HlAE3Rybinding to skeletal muscle triads was carried out in the presence of increasing concentrations of KC1 (A ), ATP ( B), and Ca2+( C )as described under "Experimental Procedures." All experiments were preformed in triplicate, and a representative example of three separate experiments is shown.
(26,371. To further characterize the interaction between ABRy and theskeletal ryanodine receptor, we performed an analysis of [3H]ABRy binding to triads in the presence of KC1 and ATP. f3H1ABRybinding to the skeletal ryanodine receptor is sensitive to changes in ionic strength as demonstrated by increased binding in thepresence of increasing KC1 concentrations (Fig. 3A). Binding was maximally stimulated %fold at a concentration of 0.5 M KC1, an effect very similar to that observed for f3H1ryanodinebinding to both triads and thepurified skeletal ryanodine receptor (2). [3H]ABRy binding to skeletal muscle triads is also sensitive to ATP, which can maximally increase binding at 4.5 mM (Fig. 3B). [3H]Ryanodinebinding to the skeletal ryanodine receptor is also stimulated by millimolar concentrations of adenine nucleotides (37). Such concentrations of ATP stimulate the rate of Ca2+release from both skeletal and cardiac muscle sarcoplasmic reticulum vesicles (22, 38) and increase the channel activity of both receptors in planar lipid bilayers (3, 22). Because Ca2+is animportant regulator of Ca2+release from
+ ++ - FIG. 4. [%lABRy photocoupling to skeletal muscle ryanodine receptor. Left, Coomassie Blue-stained polyacrylamidegel of t3HlABRy (15 nM) covalently coupled to skeletal muscle triads in the presence (+) or absence (-) of 10 ryanodine (CB);center, immunoblot stained with the affinity-purified polyclonal antibody Rabbit 46 against the COOHterminal 15-amino acid peptide of the skeletal ryanodine receptor (Rub46);right, fluorogram of a polyacrylamide gel stained with Coomassie Blue, treated with Enlightning, dried, and placed on KodakXAR-5 film for 3 weeks a t -80 "C with an intensifying screen. The arrow indicates the skeletal muscle ryanodine receptor. Molecularmass standards (inkilodaltons) are indicated to the leR.
the ryanodine receptor, we have also examined the effect of Ca2+ concentrations on the binding of [3H]ABRy to skeletal muscle triads. Ca2+increases [3HlABRy binding to theskeletal muscle ryanodine receptor at concentrations between 100 nM and 100 p~ (Fig. 3C). The half-maximal stimulation of L3H1ABRy binding occurs at -5 p ~ The . resultant bell-shaped Ca2+response curve (Fig. 3C) is very similar to the effect of Ca2+on f3H1ryanodine binding to skeletal, cardiac, and brain ryanodine receptors (26, 37). Since r3H1ABRy contains a photoactivable azido group, this compound was used to covalently label skeletal muscle triads following equilibrium binding. In the presence of W light, [3H]ABRy covalently binds to the skeletal muscle ryanodine receptor (Fig. 4, right). Covalent incorporation of[3HlABRy into the skeletal muscle ryanodine receptor was abolished with ) 4, right). The identity excess unlabeled ryanodine (10 p ~ (Fig. of the labeled protein as the skeletal muscle ryanodine receptor was confirmed by immunoblot analysis (Fig. 4, center) and by immunoprecipitation with affinity-purified polyclonal antibodies against the ryanodine receptor (data not shown). The smaller band just below the 565-kDa protein (-500 kDa) that was identified by photocoupled [3H]ABRyand affinity-purified antibodies against the ryanodine receptor is a previously recognized COOH-terminal proteolytic fragment of the receptor. Photolabeling experiments performed in the presence of magnesium and ruthenium red, at concentrations known to inhibit ryanodine binding, abolished the [3H]ABRylabeling of the skeletal muscle ryanodine receptor (data not shown). To further define the ryanodine-binding site, tryptic digestion of the [3H]ABRy-photolabeledskeletal muscle ryanodine receptor was performed to identify proteolytic fragments containing the ABRy-binding site. Fig. 5 demonstrates the appearance of a 76-kDa fragment after tryptic digestion that bound [3H]ABRy and was also identified with the affinity-purified COOH-terminal polyclonal antibody againstthe ryanodine receptor. Furthermore, a-chymotryptic digestion of the [3H]ABRy-labeled skeletal muscle ryanodine receptor also showed that theantibody staining patternof the COOH-terminal ryanodine receptor fragments was nearly identical to the pattern obtained from the fluorogram of [3H]ABRy-labeledryanodine receptor fragments (data not shown). Recently, it has been demonstrated that one of the major tryptic fragments resulting from a partial digestion of the ryanodine receptor is a 76-kDa fragment that contains the carboxyl-terminal portion of
Photoaffinity Labeling
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13079
receptor gene products. The identification of the ryanodinebinding site could also provide information on how ryanodine and other modulators of the Ca2' release channelaffect channel function and could possibly help to determine the structure of the Ca2+ channelpore.
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REFERENCES
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1. Imagawa. T., Smith, J. S.. Coronado. R.. and Campbell. K. P. (1987) J . Btol. Chem. 262,1663&16613 2. Inui. M.. Saito. A., and Fleischer. S. (1987) J. B i d . Chem. 262. 1740-1747 3. Lai, A. E. Erickson. H. P.. Rousseau. E.. Liu. Q.Y., and MetRsner. G . (1988) Nature 331. 315-319 4. Inui. M.. Satto. A.. and Fleischer. S. (1987) J . Biol. Chcm. 262. 15637-15642 5. McPherson. P. S., Kim. Y. K.. Valdivia. H.. Knudson. C. M.. Takekura. H.. Franzini-Armstrong. C.. Coronado. R.. and Campbell,K. P. ( 1 9 9 1 J Neuron 7. 17-25 6. Ellisman. M. H.. Deerinck. T. J. 0uyang.Y.. Beck, C. E, Tanbley, S.J.. Walton. P. D.. Airey. J. A,. and Sutko. J . L. ( 1 9 9 0 ) Neuron 6, 13.5146 7. Takeshima. H.. Nishimura. S.. Mataumoto. T..Ishida. H.. Knngawa. K. Mi-
of I'HlABRy-labeled skeletal muscle triads (250 pg) digested for 0, 1, 10, and 15 min a t room temperature ( C B); center, immunoblot stained with namino. N., Matauo, H., Ueda. M.. Hanaoka. M..Hirose. T.. and Numa. S. the affinity-purified polyclonal antibody Rabbit 46 against the COOH(1989) Nature 339,439445 terminal 15-aminoacid peptide of the skeletal muscle ryanodine recep- 8. Zonato. E. Fuji, J..Otau. K.. Phillips. M..Green, N. M.. Lai. E A,. Meisaner. G . . and MacLennsn. D. H. 11990) J . Bid. Chem. 266.2244-2256 tor (Rab46);right, fluorogram of apolyacrylamide gel treated with E. Green. N. M.. and MacLen. 9. Otau. K.Willard, H. E , Khanna. V.K.. Enlightning, dried, and placed on Kodak XAR-5 film for 3 weeks a t nan. D. H. (1990) J . Btol. Chem. 286. 13472-13403 -80 "C with an intensifying screen. Molecular mass standards (in kilo10. Nakai. J.. Imagawa. T., Hakamata. Y..Shtgekawa. M.,Takeshima. H.. and daltons) are indicated to the left. Numa. S. (1990) FEES Lett. 271. 169-177 11. Hakamata.Y.. Nakai. J..Takeshima, H.. and lomoto. K.(1992lFEBSlatt. 312,
Zorzato.
the receptor (39).Our data are consistent with observation this and also demonstrate that the 76-kDa COOH-terminal frag- 12. ment, which is likely to contain transmembrane domains, is 13. critical for the formation of the high affinity ryanodine-binding site. Since a known modulator of ryanodine binding, ATP, has 14. also been demonstrated tobind to a similar 76-kDa fragment of 15. the ryanodine receptor after partial tryptic digestion (27), it is 16. possible that the same76-kDa tryptic fragment that iscritical in forming the high affinity ryanodine-binding site also con- 17. tains theATP-binding site. 18. Salama et al. (40) have suggested that ryanodine also inter- 19. acts with a 106-kDa protein present in the sarcoplasmic reticu- 20. lum of skeletal muscle. In planar lipid bilayers, ryanodine was thought to affect this sulfhydryl-gated protein at concentra- 21. tions (nanomolar) that would suggest a high affinity interac- 22. tion between ryanodine and the 106-kDa protein (16). While this protein may be involved in thefunction of the sarcoplasmic 23. 24. reticulum, our data demonstrate that ABRy, which shares high 25. structural homology with ryanodine, is capableof binding only 26. to the 565-kDa ryanodine receptor/Ca2+release channel. 27. The results presented here indicate that the tritiated photo- 28. activable derivative of ryanodine, ABRy, interacts directly and 29. specifically with the active ryanodine-binding site on the Ca2+ release channel. Our results also demonstrate that the binding 30. of t3H1ABRy to the skeletal muscle ryanodine receptor is allo- 31. sterically regulated by KCI, ATP, and Ca2', compounds known to directly affect Ca2+ releasefrom the ryanodinereceptor. The 32. identification of the 76-kDa COOH-terminal tryptic fragment 33. of the skeletal muscle ryanodine receptor covalently binding 34. 35. [3HlAE5Ry illustrates that thisregion of the Ca2+ release channel is critical in the formation of the high affinity ryanodine36. binding site. Localization of the highaffinity ryanodine-binding 37. site will provide new information on the structure and func- 38. 39. tional relationship between ryanodine and the Ca2+ release channel. This information should be useful in determining if 40. this binding site is conserved among the different ryanodine
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