complex of 1.6 mCi/mg of protein, assuming complete recovery of ... storage on hard disk. .... single major polypeptide band that migrated between az mac-.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Issue of October 5, pp. 16776-16785,1989 Printed in U.S A .
Vol , 264, No.
The Ryanodine Receptor-Ca2+ Release Channel Complex of Skeletal Muscle Sarcoplasmic Reticulum EVIDENCE FOR A COOPERATIVELY COUPLED, NEGATIVELY CHARGED HOMOTETRAMER* (Received for publication, April 3, 1989, and in revised form, June 6, 1989)
F. Anthony LaiS, Manoj Misrai, LeXu, H. Amy Smith, and GerhardMeissner From the Departments of Biochemistry and Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 and the $Department of Cell Biology, Duke University Medical Center, Durham, NorthCarolina 27710
The subunit structure of the rabbit skeletal muscle tor-Ca2+ release channel complex which comprises a ryanodine receptor-Ca2+ release channel complex was homotetramer of negatively charged and allosterically examinedfollowingsolubilization of heavysarcocoupled polypeptides of M, 400,000. plasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [‘Hlryanodine binding was reThe muscle sarcoplasmic reticulum (SR)’ is an intracellular tained upon solubilization of the complex in Chapsbut membrane compartment specialized for the cyclical uptake, was lost in Zwittergent 3-14. The purified complex storage, and release of Ca2+ions (1-4). Relaxation of muscle migrated as a single peak with an apparent sedimen- results from a reduction in the myoplasmic free Ca2+concentation coefficient of -30 and -9 S upon density gradient centrifugation and with isoelectric pointsof 3.7 tration and is effected by an efficient SR Ca2+uptake and and 3.9 upon two-dimensional gel electrophoresis in storage mechanism, which is mediated, respectively, by an SR membrane Ca2+ pump (Ca,Mg-ATPase) andSR lumenal Chaps and Zwittergent 3-14, respectively.Electron Ca2+-bindingprotein (calsequestrin). Contraction is initiated microscopy of negatively stained samplesindicated that the distinct four-leaf clover structure of the ry- by the rapid massive release of Ca2+ions through Ca2+release localized regions of the SRcalled terminal anodine receptor observed in Chaps disappeared fol- channels present in lowing Zwittergent treatmentof the 30 S complex and cisternae (5-7). The terminal cisternaemembrane forms juncinstead showed smaller, round particles. Ferguson plot tional associations withothe transverse tubule (T-) system, analysis following sodium dodecyl sulfate-polyacryl- thereby creating a -120 A gap across which span large protein amide gel electrophoresis of partial and fully cross- structures that have been characterized morphologically and linkedand incompletely denatured complexes sug- variously termed feet, bridges, pillars, and spanning protein gested a stoichiometry of four M , 400,000 peptides/ (8-11). It is throughthese feet structures that T-system 30 S ryanodinereceptor oligomer. [‘HIRyanodine depolarization is believed to induce Ca2+release from SR, a binding to the membrane-bound receptor in 60 p ~ - l process commonly termed excitation-contraction ( E X ) coumM free Ca” revealed thepresence of both high affin- pling. ity (KO = 8 nM, Hill coefficient (nH)= 0.9) and low Major advances toward characterizing the Ca2+ release affinity (nH 0.45) sites witha ratio of 1:3. Reduction process have been made in recent years following the incorin free Ca2+ to 50.1 p~ or trypsin digestion of the poration of single SR Ca2+release channels into planar lipid membranes resultedin loss of high affinity but not low bilayers (12,13) and theidentification of a ligand, ryanodine, affinity ryanodine binding (HillK D= 5,000 nM, nH = which interacts specifically and with high affinity with the 0.9).Addition of 20 mM caffeine to the nanomolar Ca2+ release channel (14-19). Ryanodine, a neutral plant alkaloid, medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low induces the channel intoan open substate conductance at low affinity sites altered the rate of [‘Hlryanodine disso- concentrations(nanomolar), whereas at higher concentrations (>micromolar) itappears to close the channel comciationfromthehighaffinity sites. Singlechannel recordings of the purified ryanodine receptor channelpletely. Isolation of the SR ryanodine receptor has indicated it to incorporated into planar lipid bilayers also indicated the existenceof high and low affinity sites for ryano- be an oligomeric complex of apparent sedimentation coeffidine, occupation of which resulted in formation of a cient 30 S (20), comprising high molecular weight polypepsubconducting and completely closed state of the chan- tides of apparent relative molecular mass (MA 400,000 (21nel, respectively. These results are compatible with a 23). Reconstitution of the purified ryanodine receptor into subunit structuralmodel of the 30 S ryanodine recep- planar lipid bilayers has revealed an intrinsic Ca2+ channel activity with all the functional properties described previously * This work was supported by United States Public Health Service for the native SR Ca2+release channel (23,24).Morphological
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Grants AR18687 and GM30598 and by a Muscular Dystrophy Association postdoctoral fellowship (to F. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ The Carl M. Pearson Fellow of the Muscular Dystrophy Association (1989-90). To whom correspondence should be sent: Dept. of Biochemistry CB 7260, University of North Carolina, Chapel Hill, NC 27599-7260. Tel.: 919-966-5021.
The abbreviations used are: SR, sarcoplasmic reticulum; T , transverse tubule; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; DIFP, diisopropyl fluorophosphate; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid; Zwittergent 314, N-tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate;E-C, excitation-contraction; SDS, sodium dodecyl sulfate; KDH,Hill KO; nH,Hill coefficient; BmaX, maximal binding activity; Pipes, 1,a-piperazineethanesulfonic acid.
16776
16777
Subunit Structure of Calcium ReleaseChannel
Electron Microscopy-The 30 and 9 S protein peaks isolated from glycerol gradients (as described above) in the presence of Chaps and Zwittergent 3-14,respectively, were analyzed by negative stain electron microscopy. Aliquots (10 pl) of the 30 or 9 S peak fractions were deposited onto 400 mesh glow-discharged carbon-coated grids and washed with a few drops of 2% uranyl acetate and air dried. Images were recorded at 60,000 and 82,000 X magnification on a Philips 420 electron microscope operated a t 100 kV. Iodinution of 30 S Complex-The 30 S ryanodine receptor (25 pg) was iodinated with 200 pCi of NalZ5Ifor 30 min a t 23 "C in 0.5 ml of 0.2 M NaCl, 1% Chaps, and 20 mM NaH2POd, pH 7.0, using N chlorobenzenesulfonamide (Iodobeads, Pierce Chemical Co.). Following desalting on an Excellulose GF-5 column (Pierce), 80% of the counts inthe initial reaction mixture were removed from the protein, indicating an approximate specific activity for the radiolabeled 30 S complex of 1.6 mCi/mg of protein, assuming complete recovery of protein. SDSgel electrophoresis and autoradiography of the iodinated protein indicated the presence of a single major band at M , 400,000, with a faint band comigrating with the dye front at thebottom of the gel, presumably free lZ5I(not shown). Sedimentation on 5-20% sucrose gradients in the presence of Chaps gave a radioactive peak at 30 S, identical to that of the unlabeled ryanodine receptor (not EXPERIMENTALPROCEDURES shown), suggesting that no change in oligomeric size had occurred Materials-Ryanodine was obtained from AgriSystems Interna- due to theiodination procedure. tional (WindGap, PA), [3;H]ryanodine(54.7 Ci/mmol) from Du PontIsoelectric Focusing-A granulated gel medium for isoelectric foNew England Nuclear, and lZ5Ifrom Amersham Corp. Phospholipids cusing was prepared by swelling Sephadex IEF (50 mg/ml) in 10% were purchased from Avanti Polar Lipids Inc. (Birmingham, AL), sorbitol, 1 mM DIFP containing either 1% Chaps or 1% Zwittergent Chaps from Boehringer Mannheim, Zwittergent 3-14 from Calbi- 3-14. To 4.5 ml of the above was added 0.35 ml of either Pharmalyte ochem, and glutaraldehyde from Aldrich. SDS gel molecular weight 3-10 or Pharmalyte 2-5, and 50-100 nCi of iodinated ryanodine standards, ampholytes (Pharmalyte pH 2-5 and pH 3-10), Sephadex receptor. The mixture was incubated at room temperature for 15 min, IEF, andhydrazine hydra1;e (85%aqueous solution) were from Sigma. then poured onto a piece of Gelbond film (12 X 4 cm) placed on the All other chemicals were of analytical grade. horizontal cooling plate of a Bio-Rad 1415 electrophoresis cell. ElecPreparation of Purified 30 S ea2+Release Channel Complex-The trode strips (4 cm) prewetted in cathode (1 M NaOH) or anode (0.1 30 S Ca2+release channel complex was isolated by sucrose density M H3P04 or 0.5 M CH3C02H) solution were placed at either end of gradient centrifugation following solubilization of "heavy" SR vesicles the gel. Focusing was performed at 4 "C for 4 h using a Bio-Rad 1420 in the presence of Chaps as described previously (23). Heavy SR (2000 V) power supply at 4 watts constant power. Chaps and Zwitvesicles enriched in Ca2+release channel activity were prepared from tergent 3-14 gels containing the same ampholyte pH range (either 3rabbit skeletal muscle honnogenatesin the presence of 2.5 mM EGTA 10 or 2-5) werefocused simultaneously. Upon completion of the and 1 mM diisopropyl fluorophosphate (DIFP) by differential pellet- focusing run, each gel was cut into15 X 8-mm strips andcounted for ing a t 2,500-35,000 X g. The pellets were resuspended in 0.5 M NaCl, radioactivity in a Packard5110 y-scintillation spectrometer. The pH 20 mM N&P20,, 0.1 mM EGTA, 0.1 mM Ca2+,0.1 mM DIFP, and 20 of each strip was then determined following resuspension of the gel mM NaH2P0,, pH 7.1, and incubated for 1 h in ice. After sedimen- in 0.5 ml of deionized water, after which aliquots (0.1 ml) were taken tation at 100,000 X g for 30 min through a cushion of 0.5 M sucrose, for SDS gel electrophoresis. 5 mM K-Pipes, pH 7.0, and resuspension of the pellet in 0.3 M sucrose, Glutaraldehyde Cross-linking-The purified 30 S ryanodine recep5 mM K-Pipes, pH 7.0, membranes were rapidly frozen and stored at tor complex (8 pg in 0.4 ml of 10 mM Na-Pipes, pH 7.0, 0.5 M NaC1, -80 "C. and 1% Chaps) was mixed with 50 pl of 0.1 M glutaraldehyde and Vesicles (1.0-1.5 mg of protein/ml) were solubilized in medium A incubated a t 23 "C. Aliquots (0.1 ml) were removeda t 0,5,20, and 60 (0.9 M NaCl, 50 pM EGTA, 100 pM CaC12,5 mM AMP, 1 mM DIFP, min after glutaraldehyde addition, and unreacted glutaraldehyde was 20 mM Na-Pipes, pH 7.1) containing 1.6%Chaps, 5 mg/ml phospha- quenched with 20 pl of 10%hydrazine for 1 min. Identical experiments tidylcholine, 1 mM dithioerythritol, and 10 nM 13H]ryanodine.After using the Zwittergent-purified 9 S protein peak were performed incubation for 2 h at 23 "C!,small amounts of insoluble material were simultaneously. Samples were then mixed with 30 pl of 5% SDS, 5% removed by centrifugation for 30 min at 100,000 X g. The supernatant @-mercaptoethanol,25%glycerol, 0.25 M Tris-HCI, pH 6.8, and heated (15 ml) containing the solubilized 30 S channel complex was layered at 90 "C for 5 min before SDS gel electrophoresis (see below). Gels at the top of six 5-20% linear sucrose gradients in medium A con- were silver stained and optically scanned using a Zeineh SLR scantaining 1% Chaps and 5 mg/ml phosphatidylcholine. The gradients ning densitometer (Biomed Instruments Inc.). were centrifuged at 2 "C in a Beckman SW 28 rotor at 26,000 rpm SDS-Polyacrylamide Gel Electrophoresis-Gel electrophoresis was for 16 h followed by fractionation into 2.0-ml fractions, 0.05 mlof performed in either 5-12% or 2.3-6% polyacrylamide gradients and which was used to determine radioactivity by scintillation counting. 3 or 2.3% polyacrylamide stacking gels, respectively, in a Hoefer SE Bound [3H]ryanodinepeak fractions in the lower half of the gradient 600 vertical slab gel unit with the buffer system of Laemmli (28). The were concentrated with the use of aCentriprep 30 concentrator acry1amide:bisacrylamide ratio for all gel concentrations was 39:l. (Amicon,Danvers, MA), then diluted with 10 volumes of 0.5 M NaC1, Unless otherwise indicated, samples were denatured at 90 'C for 5 10 mM Na-Pipes, pH 7, containing 0.1% Chaps and reconcentrated. min in 2% SDS, 2% 0-mercaptoethanol, 0.1 M Tris-HC1, pH 6.8, and The last two steps were repeated to lower the percent sucrose (as 10% glycerol before electrophoresis. Gelswere either Coomassie measured with a refractometer) to below 10%. stained or silver stained by the method of Oakley et al. (29) or dried Gradient Adysis of Purified eaz+Release Channet Complex in on a Hoefer SE 540 slab gel dryer before autoradiography with Kodak Chaps and Zwittergent 3-1 4-The purified 30 S Caz+release channel x-ray (XAR-5) film at -70 "C. complex, concentrated as described above (100 pg of protein in 0.8 Single Channel Recordings-The planar lipid bilayer technique ml), was layered a t the top of 15-30% (w/w) glycerol gradients in 20 employed in these studies was essentially that used to observe the mM Na-Pipes, pH 7.0, 1.0 M NaC1, 50 p~ EGTA, 100 p~ CaC12, 5 single channel behavior of the native Ca2+release channel (12, 13). mM AMP, 1 mM DIFP, and either 0.9% Chaps or 0.9% Zwittergent Mueller-Rudin lipid bilayers (phosphatidylethanolamine, phosphati3-14. After centrifugation for 14 h at 2 "C and 28,000 rpm in a dylserine, and phosphatidylcholine in a 5 3 2 ratio in n-decane soluBeckman SW 41 rotor, fractions of 1 ml were collected and assayed tion) were formed on a circular aperture (300-pm diameter) through for radioactivity and protein content. Apparent sedimentation coef- a polyvinylidene difluoride wall separating two chambers (3-ml volficients were determined in Chaps using an enzyme marker calibra- ume each). Aliquots of purified ryanodine receptor protein (0.2-3 pl) tion curve (23) and in Zwilkergent 3-14 by centrifugation for 40 h a t were added to one side (defined as cis) of the two chambers that 2 "C and 31,000 rpm in a Beckman SW 41 rotor using aldolase (8 S) contained 20 mM K-Pipes, pH 7.0,150 p~ CaC12, 100 p~ EGTA, and and catalase (11.2 S) as sedimentation markers in separate parallel 250 mM KCl. Ryanodine receptor channels were incorporated spontaneously into the lipid bilayer and were detected as stepwise ingradients.
examination of the isolated ryanodine receptor has further revealed its striking resemblance to the feet proteins that span theT-SRjunctional gap (23,25,26). It therefore appears that the 30 S ryanodine receptor, Ca2+release channel, and feet structures areequivalent entities, which further suggests that structural,functional, and regulatory (transduction)roles are all present within a single oligomeric protein complex of 30 S. In the present report, we have further characterized the ryanodine receptor instudies that indicate that the 30 s protein complex most likely exists as ahomotetramer of negatively charged and cooperatively coupled M, 400,000 subunits. Knowledge of the subunit composition and stoichiometry may lead to insights into the mechanisms through which the receptor cclmplex exerts its apparently multiple roles during E-C coupling. Some of these results have been presented in abstract form (27).
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16778
of Calcium Release Channel
Subunit Structure
creases in current (23, 24). Electrical signals were filtered a t 300 Hz through an eight-pole low pass Bessel filter and digitized a t 2 kHz for storage on hard disk. rH]Ryanodine Binding-Unless otherwise indicated, SR vesicles (0.5-3 mg of protein/ml) were incubated with varying concentrations of free Ca2' at 22 "C in a medium containing 20 mM Na-Pipes, pH 7.0, 1 M NaCI, 1 mM DIFP, 5 mM AMP,and 2nM-3 mM ['HI ryanodine. ['HIRyanodine (54.7 Ci/mmol) was added to a concentration up to 25 nM; greater concentrationswere prepared as admixtures of labeled and unlabeled ryanodine. After 15 h, aliquots of the vesicle suspensions were (i) placed into a scintillation vial to determine total radioactivity; (ii) centrifuged for 30 min a t 90,000 X g in a Beckman Airfuge to determine free ['Hlryanodine; and (iii) placed, after dilution with 25 volumes of ice-cold water on a Whatman GF/B filter soaked in 1%polyethyleneimine. After rinsing with 3 5-ml volumes of ice-cold water, radioactivity remaining with the filters was determined by liquid scintillation countingto obtain bound['Hlryanodine. Protein Assay-Protein concentrations were determined by the method of Kaplan and Pederson (30) using Amido Black and 0.45pm Millipore filters (type HA). Bovine serum albumin was used as the protein calibration standard.
a
- 44 340 -700 4 205
4 116
4 97.4 4 66 4 45
4 29
RESULTS
Effects of Zwittergent 3-14 on PHJRyanodine Binding and Sedimentation. Behavior of the Isolated Ryanodine Receptor Complex-Fig. 1 illustrates the marked contrary effects of the addition of the zwitterionic detergents Chapsand Zwittergent 3-14 on specific ['Hlryanodine binding to preparations of heavy SR membranes. In the absence of detergent, the SR membranes bound ['Hlryanodine withacapacity of -30 pmol/mg of protein. When membranes were solubilized in a medium containing 1% Chaps and assayed for ryanodinebinding activity, both the low and high affinity sites for [3H] ryanodine were essentiallyunaltered as evidenced by the similar binding curves in the presence and absence of Chaps. However, when 1% Zwittergent 3-14 was added to the incubation medium, all measurable specific ryanodine binding was abolished. These observations of such strikingly distinct effects of two related zwitterionic detergents on specific ryanodine binding to heavy SR were further pursued by characterization of the sedimentation behavior of the Zwittergenttreated purified ryanodine receptor complex. The Chaps-solubilized ['Hlryanodine-labeled receptor was isolated, as previously described (23), as a 30 S complex comprisingpolypeptides of M, 400,000 (Fig. 2a). Sedimentation analysis of the purified ryanodine receptor complex was then performed on linear 15-30% glycerol gradients in the presence of either
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20
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10
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+ Zwittergent 3-1 0: 10-9
4 A.
10-8
10-7
10-6
10-5
TOTAL [3H]RYANODINE (M)
FIG.1. Effect of Chaps and Zwittergent 3-14on [3H]ryanodine binding to SR membranes. Heavy SR vesicles (5 mg/ml) were incubated with the indicated concentrations of ['HJryanodine for 3 h a t 37 "C in 1.0 M NaC1, 100 p M EGTA, 150 p M CaZ', 5 mM AMP, 1 mM DIFP, and20 mM Na-Pipes, pH 7.2. Labeled membranes were diluted with 0.10 volume of H 2 0 ( 0 ) .10% (w/w) Chaps (O), or 10% Zwittergent 3-14 (A) and incubated for 1 h a t 23 "C; specific ['Hlryanodine binding was determined as described under "Experimental Procedures."
$ ".-tl 10
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3
.-.-.
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9
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12
FRACTION NUMBER
FIG.2. Sedimentation profile of purified 30 S Ca2+ release channel complex in Chaps- and Zwittergent 3-14-containing glycerol gradients. a, Coomassie-stained gel of 3 pg of the purified 30 S ryanodine receptor complex electrophoresed through a 5-12% polyacrylamide gradient gel. Molecularweight standards (29,000, carbonic anhydrase; 45,000, ovalbumin; 66,000, bovine serum albumin; 97.4000, phosphorylase b; 116,000,8-galactosidase;205,000, myosin; 340,000, nonreduced a2macroglobulin; -700,000, nebulin (53)) right. The M , with correspondingM , values (XIO-') are shown to the of the ryanodine receptor has been nominally assigned as -400,000 in the text (see "Discussion"). b, the Chaps-purified 30 S ryanodine receptor complexwas sedimented through linear 15-30% glycerol gradients containing either Chaps (0)or Zwittergent 3-14 (0).Gradients were fractionated andassayed for protein content. The protein peakinZwittergent 3-14 sedimented betweenaldolase (8 S) and catalase (11.2 S) markers correspondingto an apparent sedimentation coefficient of 9 S.
Chaps or Zwittergent 3-14 followed by analysis of the radioactivity and protein content of each fraction. The resultant sedimentation profiles revealed a markedshift in the apparent sedimentation coefficient of the protein peak from 30 S in Chaps to 9 & 1 S in Zwittergent 3-14 (Fig. 26), suggesting that a dissociation of the 30 S oligomer into smaller structures had occurred. More extended centrifugation of the Zwittergentcontaining gradients revealed a clear dissociation of the 9 S proteinpeak from the [3H]ryanodine radioactivity, which remained at the top of the gradient (not shown). This suggests that as observed for membrane preparations (Fig. l), a complete loss of specific ryanodine binding to the 30 S complex had occurred following its dissociation into -9 S particles upon Zwittergent 3-14 exposure. In contrast, greater than 80%of applied radioactivity was recovered in the protein peak of glycerol gradients containing Chaps(not shown). Electron Microscopy of the Ryanodine Receptor Isolated in Chaps or Zwittergent 3-l4"Structural analysis by electron microscopy was performed on negatively stained samples of the Chaps-purified 30 S complex and on the 9 S protein isolated from the Zwittergent 3-14 gradients. As observed previously (23), the Chaps-solubilized ryanodine receptor isolated by density gradient centrifugation displayed the characteristic four-leaf clover (or quatrefoil) morphology (Fig. 3a) similar to that described for the shadowed images of the T-
FIG. 3. Electron microscopy of the 30 a n d 9 S particles. Negative stain electron microscopy was performed on the protein peak fractions isolated from Chaps ( a ) and Zwittergent ( b and c) gradients. a, three selected images of the quatrefoil structure of the 30 S ryanodine receptor-Ca2+release channel complex. b, a field of the isolated 9 S particles derived from centrifugation of 30 S quatrefoils in a Zwittergent 3-14-containing gradient. c, selected images of the 9 S particles. Scale bars represent 200 A.
SR-spanning feet structures (31). The quatrefoil dimensions are 34 nm from the tipof one leaf to thatof the opposite one, with each leaf 12-14 nm in diameter. Each leaf appears to enclose a stain-filled depression or cavity of 2-4 nm. At the center of the complex is a 14-nm diameterprotein-dense region surrounding a small central hole of 1-2-nm diameter. In contrast to the distinct cloverleaf structures observed in Chaps, however, the images of the 9 S particles obtained following Zwittergent 3-14 treatment of the 30 S complex were completely devoid of quatrefoils and instead showed smaller, fairly uniformly round shaped particles(Fig. 3b). The average size of the 9 S particles was 8.5 k 2 nm, a value less than the12-14-nm diameter of each leaf of the 30 S quatrefoil structure. However, it was notable that a significant fraction of the dissociated subunits appeared to have an accumulation of stain in the center (Fig. 3c), suggesting the possible presence of a small central hole or depression. This observation is somewhat reminiscent of the similar features of stain accumulation found in the leaves of the intactquatrefoil (Fig. 3a). Thus, itis not inconceivable that thesmall hole or depression in the 9 S particles may have in some way derived from a collapse of the leaves of the quatrefoil. In supportof the latter suggestion, time course studies in which the 30 S quatrefoils in Chapswere incubated withan equal volume of 1%Zwittergent 3-14 (final concentrationof 0.5%)revealed an immediate shrinking of the depressions in the four leaves of the cloverleaf structure within 1 min after mixing. This resulted in areduced quatrefoil size of -27 nm from tip to tip,compared with the 34 nm regularly observed (not shown). In samples taken from subsequent time points (>5 min), structures recognizable as quatrefoils were completely absent. Isoelectric Focusingof the l'2511Ryanodine Receptor in Chaps and Zwittergent 3-14-SDS-polyacrylamide gel electrophoresis of the purified 30 S complex resolved the protein into a single major polypeptide band thatmigrated between azmacroglobulin (Mr340,000) and nebulin ( M , 700,000) with an M , of -400,000 (Fig. 2a). To address the possibility of subunit heterogeneity of the M , 400,000 polypeptide the electropho-
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reticproperties of theintact complex and its dissociated subunits were further determined by radiolabeling the 30 S complex with '1 and subsequent electrofocusing. Hence, isoelectric focusing in Sephadex IEF was performed under dissociating and nondissociating conditions in the presence of either Zwittergent 3-14 or Chaps, respectively, and using ampholytes in the pHranges 3-10 (not shown) or 2-5 (Fig. 4, u and b). Upon electrofocusing in both the pH 3-10 and 2-5 ranges, the radiolabeled 30 and 9 S particles migrated toward the anode into one major peak with similar isoelectric points. Analysis of the pH of each eluted gel slice after focusing in the pH2-5 ampholyte medium allowed a direct determination of the isoelectric point (PI) of each radioactive peak and resulted in PI values for the 30 S complex and 9 S particles of 3.7 f 0.40 and 3.9 f 0.35, respectively. The slightly more acidic PI obtained in Chaps detergent was a consistent phenomenon as bothChaps and Zwittergent 3-14 gels werealways focused simultaneously. This difference in PI maybe the result of subtle conformationaldifferences in protein secondary structurecaused by dissociating the native quatrefoil into its component subunits. SDS-polyacrylamide gel electrophoresis of the PI 3.7 and 3.9 radioactive peaks again revealed a single M , 400,000 band, indicatingthat minimal proteolytic alteration of the polypeptide had occurred during the isolation, radiolabeling, and electrofocusing procedures (Fig. 4c). These studies, which show molecular size and electrophoretic homogeneity of the dissociated subunits, suggest an absence of heterogeneity of the M, 400,000 polypeptides, implying least very that the constituents of the 30 S complex are at the similar, if not identical, subunits. Glutaraldehyde Cross-linking of the 30 S Ryanodine Receptor-Further identification of the stoichiometry of the subunits within the purified 30 S complex was approached through cross-linking studiesperformed using glutaraldehyde as the covalent cross-linking agent. Fig. 5a shows that upon addition of glutaraldehyde to a sample of the 30 S complex for various times followed by denaturing gel electrophoresis, additional bands of higher M , than the parent (top truce)
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Subunit Structure of Calcium Release Channel
16780
a
I I
4
12
16
FRACTION NUMBER
FRACTION NUMBER -
p
7
4 -700 4 340 4 205
1
116
97.4 66 45 7
29
FIG. 4. Isoelectric focusing of the 30 and 9 S particles. The ryanodine receptor was radioiodinated and electrofocused in a granulated gel medium containing ampholytes, pH 2-5, in the presenceof either Chaps (a) or Zwittergent 3-14 (b) as described under “Experimental Procedures.” The gel was cut into strips and analyzed for radioactivity (0)and pH ( X ) . The PI values determined in Zwittergent 3-14 and Chaps were 3.9 0.35 and 3.7 0.4, respectively ( n = 6). c, autoradiogram of the PI 3.9 (lane Z ) and 3.7 ( l a n e C) peaks after SDS gel electrophoresison a 5-12% polyacrylamide gradient gel. areshown tothe right as Molecular weight markers (M,X descrihed in the Fig. 2 legend.
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*
%T
d
-400,000 subunit became apparent. Increased time of exposure to thecross-linking agent resulted in greater accumulation of the M , > 400,000 bands (second and third traces) 4 resulting eventually in a single high molecular weight band close tothetop of the gel (bottom trace). Densitometric scanning of the gel revealed three major species that could be FIG. 5. Glutaraldehyde cross-linking of 30 and 9 S ryanoresolved in addition to the -400,000 protein, whose staining dine particles. Densitometric scansof silver-stained SDS intensity diminished as thehigher M , bands increased. Con- gels ofreceptor with the 30 S (a) and 9 S (b)ryanodinereceptortreated trol experiments inwhich the Zwittergent-purified 9 S protein glutaraldehyde for various times and electrophoresed through a 2.3peak was subjected to identical cross-linking conditions re6% polyacrylamide linear gradient gel (2 pgllane) asdescribed under 400,000 (Fig. 5b), “Experimental Procedures.” a, top trace illustrates asinglemajor sultedin onlyasingle band at M , indicating that intersubunit cross-linkingwas occurring spe- band ( I ) of M , 400,000 obtained at the0 min time point. The second 9 S protein. trace reveals the presenceof three additional species of M,> 400,000 cifically in the intact30 S complex but not in the (2,3, and 4 ) after a 5-min exposure with glutaraldehyde, all of which Also, sedimentation of the fully cross-linked 30 S complex increase in intensity after 20 min (thirdtrace). The fourth traceshows (see Fig. 5a, bottom trace) in Zwittergent- and Chaps-contain-complete accumulationof protein into band 4 after a 60-min exposure. ing gradients revealed a single protein peak corresponding to The arrow and double arrow correspond to the positions of a2 macthat of the regular 30 S peak in Chaps, and SDS-polyacryl- roglobulin (340,000) and nebulin (-700,000). Top indicates the interamide gel electrophoresis of the 30 S peak fraction gave a gel face between the stackinggel (2.3% polyacrylamide) and the separatprofile identical to that inFig. 5a, bottom trace (not shown). ing gel. b, identical experiment to a but using 9 S particles. A single traces. Thetimes of glutaraldehyde band ( I ) isseeninallfour Due to the large sizeof these protein bands,precise molecular exposure are as indicatedfor a. The arrow, double arrow, and top are weights could not be accurately determined by extrapolation as defined for a. c, modified Ferguson plot analysis (42.43) in which of presently available molecular weight standards, although the bands 1-4 seenin a (second and third traces) areplottedas
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16781
Subunit Structure of Calcium Release Channel +30 JJM Ryonodine
. .. e
e
4
m
.
9
+2 mM Ryonodine C-
0-
FIG. 6. Effect of ryanodine on a single reconstituted purified ryanodine receptor channel.Single channel recordings of K+ current of purified ryanodine receptor protein incorporated into a planar lipid bilayer in symmetric 250 mMKC1 buffer (20 mM KPipes, pH 7.0, 150 FM CaC12, 100 WM EGTA, 250 mM KCl) with 50 p~ free Ca2+.Unitary conductance, 700 picosiemens; holding potential, 20 mV. The upper trace shows the appearance of the subconducting state with an open probability of -1, several minutes after cis addition of 30 p~ ryanodine. An additional, infrequently observed, substate can also be noticed. The lower trace illustrates the sudden transition from the subconductance state toa fully closed state within 1 min after cis addition of 2 mM ryanodine. Bars on the kft represent the open (0)and closed ( c ) channel.
-
the M , > 400,000 bands are clearly >M, 700,000 (Fig. 5a). However, the most reasonable interpretation of the specific appearance of progressively higher M , bands on the gel upon glutaraldehyde cross-linking of the 30 but not the 9 S protein is that they represent multimers of the M, 400,000 subunit formed by the creation. of covalent cross-linking bonds between individual members of an oligomeric complex. The observation of three bandsarising from a single precursor M , 400,000 polypeptide suggests a minimum stoichiometry of four subunits present in the 30 S oligomer. This possibility was corroborated by Ferguson plot analysis of the assumed copy number uersus the relative mobility of each of the bands observed upon cross-linking of the 30 S complex (Fig. 5c). The above assumption of bands I through 4 (Fig. 5a) corresponding to monomer, dimer, trimer, and tetramer, respectively, produced a linear graph with correlation coefficient, r = 0.97, suggesting the probable existence of a direct stoichiometric relationship among bands 1 through 4 . Further, compatible with the above suggestions, an interestingsimilar observation of three bands of M, > 400,000 in addition to the major M, 400,000 monomer could also be obtained when the purified ryanodine receptor complex was mixed with SDS sample buffer without heating immediately prior to electrophoresis (Fig. 5d). Ferguson plot analysis of thesebands produced a lineargraph.similar to thatin Fig. 5c (not shown). Ryanodine Binding--Ryanodine has been shown to bind with high affinity and in a Ca2+-dependentmanner to theSR Ca2+ release channel (reviewed in 32). Fig. 6 shows that
-
-
-
~
~
~~
'O'O
1 .o
I
0.1
0.01
0.001 10-l~
10-8
10"
FREE [3H]RYANOOlNE (U)
FIG. 7. Dependence of [3H]ryanodine binding on Ca"* concentration. Heavy SR vesicles (3 mg of protein/ml) were incubated for 15 h a t 22 "C in a medium containing 20 rnM Na-Pipes, pH 7.0, 1.0 M NaCl, 1 mM DIFP, 5 mM AMP, 2 nM-3 mM [3H]ryanodine,
and either lo" M free Ca2*(1 mM Ca2+and 5 mM EGTA), 2 X M free Ca2' (1mM Ca2+and 1mM EGTA), or 1 X M free Ca2+(2 mM Ca2+and 1mM EGTA). Total bound and free [3H]ryanodinewas determined as described under "Experimental Procedures." In parallel experiments, it was established that in each condition, equilibrium of [3H]ryanodine binding was obtained. Specific binding is shown in a and in the form of Scatchard (inset of a) and Hill (b) plots and was obtained by assuming that nonspecific [3H]ryanodinebinding is linear with ryanodine concentration. Nonspecific binding was defined as the difference between total and specific binding and amounted to 22 nmol/mg of protein at 3 mM [3H]ryanodine. Hill plots were obtained assuming that specific binding of [3H]ryanodine amounted to 32 pmol/mg of protein (one high and three low affinity binding sites at 1mM Ca2+).Data shown are from one representative experiment done in triplicate. The standard errors were +-lo%or less.
~
assumed copy numbers of the parentmonomer (I) versus total acrylamide concentration (%T?. %T was plotted on a logarithmic scale (42) due tothe use of .a linear gradient gel (2.3-6%) for these of bands 1-4 experiments and was calculated from the mobility (RF) (a, second and third traces) relative to the bromphenol blue tracking dye. The correlation coefficient ( r ) of the straight line drawn through the four points obtained was 0.97. d, top portion of a silver-stained gel followingSDS-polyacrylamide gel electrophoresis of purified 30 S ryanodine receptor (0.8 p.g) partially denatured by the addition of SDS denaturing buffer (see "Experimental Procedures") immediately prior to electrophoresis at 5 "C on a linear 2.3-6% polyacrylamide gradient gel. The RF values of bands 1-4 correspond to bands 1-4 in a (second and third traces').
occupation of the high affinity site for ryanodine correlates reasonably well with the formation of an open subconducting state of the channel. A single ryanodine receptor channel was obtained by reconstituting the purified 30 S complex into a planar bilayer, under conditions in which the single channel conductance was 700 picosiemens with monovalent cations (250 mM K') as the current carrier (Fig. 6). Upon addition of W M ryanodine cis, the channel entered into a characteristic subconductance state (18,19), which had -40% of the normal conductance and an open probability close to unity (upper truce). A relatively high ryanodine concentration of 30 W M
16782
Subunit Structure of Calcium ReleaseChannel TABLEI Effect of ea2+concentration, caffeine, and trypsin digestionon pH]ryanodine binding to heavy SR vesicles [3H]Ryanodine binding parameters were determined as described in the legend of Fig. 7 for heavy SR vesicles incubated at 10-9-10-7 M free CaZ+in the presence ( n = 3) and absence (n = 5) of20mM caffeine or 5 X 10-5-10-3 M free Ca2+(n = 8). 13H]Ryanodinebinding to trypsin-digested vesicles (15 pg of trypsin/ml for 30 min at 5-10 M free Ca2+. mgof SR protein/ml; Ref. 49) was determined at 5 X aO-' M Ca2+ ( n = 5)
Binding
Low affinity &ax (pmol/mg) 5,000 KDH(nM)
32 f 12 1,000f 2,000 0.9 0.8 f 0.15
nH
C
W M Ca2+
5x
5x
M Ca2+ +trypsin (n = 3)
+ caffeine
10-~-10-~ M CaZ+ ( n = 8)
None
8f2 7f2 0.9 f 0.1
None
33 f 12 f 200 0.45 f 0.1
21f9
34 f 9 20,000 f 10,000 0.85 f 0.1
( n = 3)
f 0.2
that ryanodine binding was dependent on free [Ca"] to a greater extent at nanomolar than at micromolar concentrations. Scatchard plot analysisof the binding data obtained in 0 lOOld.4 c.2+ a 0 + 200 @ RYANOOINE 1 mM Ca2+medium indicated the presence of a high affinity P 1.5 site with a Elrnax of 8 pmol/mg of protein and KO of 6 nM (inset, Fig. 7u).Reduction of free Ca2+to 20 p~ in thebinding medium decreased the affinity of ryanodine for its high affinity site by 2-fold. Further reduction of Ca2+to 0.1 p~ and lower resulted in the loss of high affinity ryanodine binding. The data of Fig. 7a also revealed the existence of additional ryanodine-binding sites of lower affinity. The B, values for the high and low affinity sitesin thepresence of 50 pM-1 mM n z Ca2+ were 8 and 21 pmol/mg of protein, respectively (Table 3 0 I), suggesting that for each high affinity ryanodine-binding m 0.0 -" site, there are two to three additional sites of lower affinity. 0 1 2 3 4 5 The combined B,,, of high and low affinity sites (29 pmol/ TIME (h) mg) in 50 pM ca2+was similar to that of total low affinity binding sites (32 pmol/mg of protein) found at submicromolar FIG. 8. Rate of [3H]ryanodine dissociation in the presence concentrations of Ca2+(Table I). These data suggest that at and absence of 200 p~ ryanodine. Heavy SR vesicles (5 mgof protein/ml) were incubated for 3 h at 37 "C in a medium containing low Ca2+concentrations, only low affinity sites for ryanodine 20 mM Na-Pipes, pH 7,1.0 M NaCl, 1 mM DIFP, 5 mM AMP, 100 exist, whereas increasing Ca2+ to greater than micromolar p~ EGTA, 200 pM Ca2+,and 10 nM [3H]ryanodine. [3H]Ryanodine concentrations reveals apopulation of high affinitysites dissociation was initiated by diluting membranes at 37 "C 20-fold which, proportionally, represent approximately one-quarter into media containing 20 mM Na-Pipes, pH 7, 1.0 M NaCl, 0.1 mM DIFP, 5 mM AMP, and either 100 p M free Ca2+,100 p M free Ca2+ of the total number of sites obtainable. Fig. 7b shows a Hill plus 200 p M ryanodine, 0.05 p M free Ca2+(200 p M ca2+plus 1.8 mM plot analysis of the binding data obtained in 0.1 pM, 20 pM, EGTA), or 0.05 p~ free Ca2+plus 200 pM ryanodine. Bound [3H] and 1 mM Ca2+. At 0.1 p~ Ca", the data points could be ryanodine was determined as described under "Experimental Proce- approximated by a straight line with an average Hill coeffidures." of 5,000 nM. At higher cient (nH)of 0.8 and Hill KO (KDH) Ca2+concentrations, the Hill plotsshowed a nonlinear behavwas used to accelerate the otherwise very slow modification ior over the nanomolar to micromolar ryanodine concentraof the channel by ryanodine (16). Upon further addition of tion range. In the nanomolar ryanodine range, n H was close cis-ryanodine to millimolar concentrations, the channel's sub- to unity (0.9), implying an absence of allostery between occonductance state abruptly disappeared, and the channel en- cupied high and unoccupied low affinity sites. At higher tered into a fully closed state (lower truce). This appeared to concentrations, however, the nH decreased to 0.45, suggesting be a ryanodine-specific effect as the channelremained closed that ryanodine binding to the other sites of lower affinity for the duration of the experiment and was recalcitrant to cis appeared to display a negative cooperativity. When the Ca2+addition of channelactivators such as Ca2+ and adenine releasing drug caffeine was added to 50.1p~ Ca2+media, the nucleotide (7). The observation of two distinct effects of low KDHwas reduced to 1,000 nM, indicating a 5-fold increase in and high ryanodine concentration on the channel's conduct- binding affinity, although the Hill coefficient (nH = 0.9) was ance behavior suggested that these effects were mediated by not appreciably affected (Table I). A similar linearity in the the sequential occupation of separate ryanodine-binding sites Hill plot could also be seen at higher Ca2+ (50 pM) after on the release channel protein. membranes were treated with trypsin (nH = 0.85). In these This observation led us to a study of the high and low proteolyzed samples, KDHwas20,000 nM (Table I). Thus, affinity binding of [3H]ryanodineto heavy SR vesicles. In all trypsin digestion of the ryanodine receptor appears to destroy experiments, 5 mM AMP was added since the presence of the negative cooperativity in ryanodine binding to the low adenine nucleotide decreases the time required to achieve affinity sites. Fig. 8 illustrates that binding of ryanodine to the low equilibrium binding at low [Ca"] (not shown). Fig. 7u shows ._ &d
2
I
I \
0.05 @ Co2+
0
"
'I
+
200pM RYANOOINE
Subunit Structure of Calcium Release
Channel
16783
muscle RNA blots have suggested a transcript size of-15 kilobases, corresponding to a predicted protein molecular weight of approximately 550,000, assuming an average amino acid molecular weight of 110.' However, an absolute value for the polypeptide mass awaits the elucidation of the complete amino acid sequence of the ryanodine receptor subunits. The present study strongly supports our earlier supposition of a tetrameric arrangement and further indicates that the subunits of the complex are identical negatively charged components that exhibit an allostery for ryanodine binding. The initial findingthat led to thisseries of studies was that of specific loss of high and low affinity ryanodine binding to SR in thepresence of the detergent, Zwittergent 3-14 (Fig. 1). The previous report of immunoaffinity isolation of the skeletal muscle SR high molecular weight proteins by Kawamoto et al. (33) also utilized Zwittergent 3-14 to solubilize the membranes, although no ryanodine-binding studies were included. Our data suggest that the 30 S ryanodine receptor complex in Chaps is dissociated into smaller 9 S particles upon exposure to Zwittergent 3-14 and results in a loss of ryanodine binding (Figs. 1-3). This specific effect may be due DISCUSSION of the interaction of the complex with the highly The high molecular weight proteins of terminal cisternae to the nature flexible hydrophobic CI4 tail of Zwittergent 3-14, since the SR were first purified and immunolocalized by Kawamoto et hydrophilic zwitterionic sulfobetaine moiety is identical to al. (33,34) in effortsto characterize the molecular components which in contrast possesses a less that span the T-SR junctional gap of skeletal muscle. More that presentinChaps, recently, solubilization of the skeletal SR ryanodine receptor flexible hydrophobic bile acid skeleton characteristic of digiin buffers containing detergent and high salt (35) and subse- tonin and thecholate seriesof detergents. A greaterapparent quent isolation by immunoaffinity chromatography (21, 24, denaturing effect of Zwittergent detergents, relative to Chaps, 36), sequential column chromatography (22), and density on protein structure and function has been reported previgradient centrifugation (20, 23) have shown it to comprise a ously (38, 39). The chemical difference in the hydrophobic groups attached to the sulfobetaine moiety is further reflected single major high molecular weight protein band of M , by the distinctive aggregation numbers of 83 and4 and critical 350,000-450,000 (nomin.ally designated here as -400,000) in micelle concentrations of 0.1 and 5 mM for Zwittergent 3-14 the form of an oligomeric complex of apparent sedimentation and Chaps (in 0.1 M Na+), respectively (40). In preliminary coefficient 30 S (20,23). The30 S ryanodine receptor complex studies, we found that theChaps-solubilized ryanodine recepfunctionally encompasses the SR Ca'+ release channel, as tor, when transferred intobuffers containing either Triton Xillustrated by its characteristic behavior when incorporated 100, Lubrol PX, MEGA-9, cholate, digitonin, or OCtyl-@-Dinto planar lipid bilayers (23, 24), and negatively stained glucopyranoside resulted in retention of high affinity ryanospecimens morphologicdly resemble the SR feet structures dine binding without dissociation of the complex into 9 S found specifically at T-SR junctions(23, 25, 26). Thus, Ca2+ particles (Fig. 2b). This is particularly interestingsince Lubrol release from the SR during E-C coupling in skeletal muscle PX, a nonionic polyoxyethylene (like Triton X-loo),MEGAappears to be mediated by a large multimeric transmembrane 9, a glucamide, and octyl-@-D-glucopyranoside,an alkyl gluprotein complex that ph,ysicallytraverses the T-SRjunctional coside, all contain hydrophobic straight chain hydrocarbon gap, across which the as yet undefined physiological trigger tails ofClz,Cg, and Ca, respectively. A more comprehensive for Ca2+release is believed to occur. Attempts toassemble all study of high affinity ryanodine binding to SR membranes the pertinentpublished data intoa plausible molecular mechsolubilized using a wider variety of detergents including the anism for E-C coupling would begreatly facilitated by a better Zwittergent 3-8 to Zwittergent 3-16 series could therefore understanding of the subunitstoichiometry, interactions, and enhance our understanding of the interactions of detergent composition of the key players involved in the excitation with the subunits of the ryanodine receptor complex which signal transduction process, of which the Ca'+ release channel result in their disaggregation. is believed to be a clear candidate. Based on our previous Negative stain electron microscopy of the dissociated 9 S observations of high affinity ryanodine-binding stoichiome- particles revealed mainly round shaped structures with an try, sedimentation properties, and morphological evidence, average diameter of 8! f 20 8, (Fig. 3). This value is similar the oligomeric structure of the 30 S ryanodine receptor-Ca2+ to that of 93 f 14 A obtained for the negatively stained release channel complex was proposed to be a tetramer of M , Zwittergent 3-14-purified high molecular weight proteins iso400,000 subunits (231. Other initial estimates of the stoi- lated by Caswell and co-workers (33). The dimensions obchiometry of the ryanocline receptor suggested it to comprise tained fo; the 9 S particles are noticeably smaller than the a dodecamer of M , 360,000 subunits (22). However, recent 120-140 A size measured for the diameter of each leaf of the physical evidence that favors a tetrameric assembly of sub- intact quatrefoil. However, the often visible presence of a units for the ryanodine receptor has come from studies using small (-10 A) central stain-filled depression in the 9 S parscanning transmissionelectron microscopy, which resulted in ticles, assuming the 9 S particles actually are derived from a value of about 2 million for the mass of the purified ryano- each leaf of a 30 S quatrefoil, suggests that there has occurred dine receptor in glutaraldehyde-fixed and -unfixed states (37). a shrinking of the volume occupied by the protein leaf upon This value would increase the size of each subunit within a treatment with Zwittergent. This reduction in size could be tetramer to M , 500,000 (26). In accord with this estimate, conceptually visualized as arising from a loss of the intersubrecent studies using isolated cDNAs encoding portions of the rabbit skeletal muscle ryanodine receptor to probe skeletal F. A. Lai, unpublished studies.
affinity sites markedly affects the rate of [3H]ryanodine dissociation from the high affinity site. A[3H]ryanodine concentration of 10 nM was used to prelabel the high affinity ryanodine-binding site initially. The ryanodine-labeled membranes were then diluted 20-fold into four media containing either (i) 0.05 p~ free Ca'+; (:ii) 0.05 p~ free Ca'+ plus 200 p~ unlabeled ryanodine; (iii) 100 p M free Ca2+;or (iv) 100 p M free Ca'+ plus 200 p M r,yanodine. In the absence of 200 p M ryanodine, [3H]ryanodinebinding rapidly decreased with time in the nanomolar Ca2+ medium (Fig. 8). In the 100 pM Ca'+ medium, which favors high affinity binding, only about half of the bound [3H]ryanotline rapidly dissociated, presumably because a new equilibrium between bound and free [3H] ryanodine was reached. However, when 200 p M ryanodine was added to both the 0.05 and 100 p~ Ca'+ dilution media, the rates of [3H]ryanodine dissociation were greatly reduced. These observations provide further direct evidence for an allosteric interaction between the high and low affinity ryanodine-binding siteson the Ca2+release channel complex.
-
-
16784
Subunit Structure
of Calcium Channel Release
unit structural constraints that occur during dissociation of the 30 S quatrefoil, which results in the consequent collapse of the protein constituting each leaf to reduce effectively the ;0-40 A depressions present in the native quatrefoil to -10 A. A partial separation of the complex into four parts hasalso been reported in rotary-shadowed images of the digitoninpurified ryanodine receptor isolated by immunoaffinity chromatography (41). Isoelectric focusing of the 30 S complex and 9 S particles has revealed the highly acidic nature of the ryanodine receptor subunits with PI values of 3.7 and 3.9, respectively (Fig. 4). The nature of the subtle differences of PI inChaps and Zwittergent 3-14 is unknown but may bepostulated to be due to the differential effects of each detergent on the protein conformation of the 30 S complex and 9 S particle. Analysis of the amino acid composition of the purified high molecular weight proteins (33) revealed that of the totalcharged amino acids present, 70% were acidic (41% glutamic acid and 29% aspartic acid) and 30% were basic (12% lysine and 18% arginine), giving a 2.3-fold excess of negatively versus positively charged residues. The relative proportion of charged (positive plus negative) residues was 35% of the total, although methionine andtryptophan were not determined. Thus, a quarter (24%)of the totalamino acids analyzed were found to be acidic residues. The abundance of acidic residues correlates very well with the low PI values for the ryanodine receptor and itssubunits determined in thisstudy. The glutaraldehyde cross-linking studies (Fig. 5) suggest a minimum subunit stoichiometry of 4 units per 30 S ryanodine receptor complex. A lack of cross-linking among components electron of the 9 S protein peak indicates, as is apparent in the microscopy images (Fig. 3), that the 30 S tetramer had dissociated into monomers upon exposure to Zwittergent 3-14. Furthermore, recentrifugation of the fully cross-linked 30 S protein in Zwittergent-containing gradients did not dissociate the oligomer into 9 S monomers, as wouldbe expected of covalently linked units within acomplex. These studiestherefore confirm the drastic structural effects of Zwittergent on the quatrefoil as seen in Fig.3, and the linearity of the Ferguson plot (Fig. 5c)further indicates that thecross-linked products obey a simple stoichiometric relationship conforming to dimers, trimers, and tetramers of a single polypeptide (42,43). Theappearance of the same three bands (Fig. 5d, 2, 3, and 4 ) in addition to the major M , 400,000 monomer (Fig. 5d, I ) on gels of partially denatured 30 S complexes further supports our interpretation that the cross-linking data indicate the tetrameric nature of the 30 S oligomer. Similar chemical cross-linking studies on other membrane proteins include those of Chadwick et al. (43), who showed a novel hexameric form of the Neurospora crassa plasma membrane H+-ATPase using glutaraldehyde, and Lo et al. (44), who observed a fully cross-linked species of M, 270,000 and an additional M, 95,000 component for the purified cat muscle nicotinic acetylcholine receptor using dimethylsuberimidate. The characteristics of ryanodine binding to heavy SR membranes have been studied by a number of laboratories. An initial reportby Pessah et al. (15) showed that [3H]ryanodine, synthesized by reduction of 9,21-didehydroryanodine (45), bound to skeletal muscle SR membranes with high affinity. Specific ryanodine binding was dependent on Ca2+and was optimal at -70 PM free Ca2+.Further studies have shown that heavy SR membrane preparations can specifically bind up to -20 pmol of [3H]ryanodine/mg of protein and with a K D of 4-200 nM (14,15,17, 23). In addition to theCa2+dependence of ryanodine binding, the affinity and binding capacity have been reported to be affected by adenine nucleotides, caffeine,
-
M P , polycationic dyes, and ionic strength (35, 46-48). In this study, we have demonstrated novel observations of an allosteric interaction between, and interconversion of, low and high affinity ryanodine-binding sites revealed upon raising free Ca2+from nanomolar to micromolar concentrations (Fig. 7). The observation of a changein the rate of dissociation of ryanodine from the high affinity site upon occupation of the low affinity sites further substantiated the presence of allosteric behavior between these sites (Fig. 8). The effect of ryanodine binding to the low affinity sites following occupation of the high affinity site appears to result in conformational changes that partially occlude the dissociation of the [3H]ryanodine bound to the high affinity site. Conversion of high to low affinity ryanodine binding was also observed upon prior trypsin digestion of heavy SR membranes (Table I and Ref. 49), presumably due to structural rearrangement of the high affinity site for ryanodine, and also resulting in aloss of binding cooperativity between the sites. The presence of caffeine in the nanomolar Ca2+ medium decreased the K D H of ryanodine binding from 5,000 to 1,000 nM (Table I). Caffeine, which is believed to mimic the activating effects of Ca2+ ions on SR Ca2+release (5,50,51), appeared to increase the binding affinity without restoringbinding cooperativity since the Hill coefficient was unaffected ( n =~0.9). Hence, these studies do not support previous suggestions that caffeine activates the SR Ca2+release channel by increasing the affinity of the Ca2+activating sites for Ca2+(5, 51). The observed ratio of low to high affinity sites for ryanodine in these studies is approximately 3:l (Table I), further supporting the notion of a tetrameric association of M , 400,000 subunits into a 30 S oligomer (32). The single channel recordings of Fig. 6 serve to illustrate the separate functionaleffects on channelconductance following exposure to low and high concentrations of ryanodine. The substate conductance of the channel, with -40% of the native conductance, which is reproducibly observed upon addition of micromolar cis-ryanodine (18, 19, 23, 24, 36, 52), is likely to be the consequence of ryanodine interacting with the high affinity site, which has a stoichiometry of 1 per 30 S tetramer (Fig. 7 and TableI). The subsequent complete closing of the channel by addition of millimolar cis-ryanodine can be interpreted as being due to occupation of the low affinity sites, which have a stoichiometry of 3 per 30 S tetramer (Fig. 7 and Table I). Whether the fully closed state of the channel requires occupation of the high affinity plus one, two, or all three of the low affinity sites or even other combinations cannot be discerned from the present data. Therecordings do show, however, that there are at least two clearly defined functional effects of ryanodine on the release channel. This phenomenon can be plausibly attributed to the existence of allostery among four ryanodine binding sites on the homotetramer (Figs. 7 and 8). Recent single channel recordings of the purified 30 S complex have shown the presence of multiple conductance states within the oligomer’s intrinsic channel (24, 52). Up to four approximately equivalent conductances were observed with either monovalent or divalent cations as the permeant ion. These studies were interpreted as being due either to the presence of a multibarreled oligomeric channel with each of four subunits comprising an individual conducting pore or a single conducting pore present within a tetrameric assembly 400,000 subunits (52). In both cases, cooperative of M , interactions among subunits must be postulated to occur in order to account for the appearance of the four conductance states. The resultsobtainedin this study provide further credence for the presence of intersubunit interactions within
-
-
Subunit Structure of Channel Calcium Release
16785
24. Smith, J. S., Imagawa, T., Ma, J., Fill, M., Campbell, K. P., and Coronado, R. (1988) J. Gen. Physiol. 92,l-26 25. Saito, A., Inui, M., Radermacher, M., Frank, J., and Fleischer, S. (1988) J. Cell Biol. 107, 211-219 Acknowledgments-We are grateful for the technical assistance of 26. Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Inui, M., Andrea Denk, Suzanne Goodman, Phil Hess, and Jeffrey K. LaDine. and Fleischer, S. (1989) Nature 3 3 8 , 167-170 27. Lai, F. A., Smith, H. A,, and Meissner, G . (1989) Biophys. J. 55, Note Added in Proof-Following submission of this manuscript, 207a (abstr.) Takeshima et al. (54) reported the primary structure and expression 28. Laemmli, U. K. (1970) Nature 227,680-685 of complementary DNA of the rabbit skeletal muscle ryanodine 29. Oakley, B.R., Kirsch, D.R., and Morris, N. R. (1980) Anal. receptor. Their dataindicate that theryanodine receptor polypeptide Biochem. 105,361-363 comprises a sequence of 5037 amino acids, with a relative molecular 30. Kaplan, R. A., and Pederson, P. L. (1985) Anal. Biochem. 150, mass of 565,223 and 40% more negative than positively charged 97-104 residues (736 uerses 525).Expression of the cDNA in Chinese hamster 31. Ferguson, D. G., Schwartz, H. W., and Franzini-Armstrong, C. ovary cells resulted in appearance of high affinity [3H]ryanodine (1984) J. Cell Biol. 99, 1735-1742 binding activity. These recent findings, along with the estimated M, 32. Lai, F. A., and Meissner, G. (1989) J. Bioenerg. Biomembr. 2 1 , of 2,300,000 for the ryanodine receptor oligomer (37), complement 227-246 our assertion in this paper that the 30 S ryanodine receptor-Ca2+ 33. Kawamoto, R. M., Brunschwig, J. P., Kim, K. C., and Caswell, release channel comprises an acidic, homotetrameric complex (total A. H. (1986) J. 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the 30 S oligomer, although they cannot distinguish between the two models of ion co.nduction proposed previously (52).
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