CeN CaMurn (1692) 13, 635-647 0 Longman Group UK Ltd 1992

Ryanodine-aff inity chromatography purifies 106 kD Ca2’ release channels from skeletal and cardiac sarcoplasmic reticulum G. SALAMA’, M. NIGAM’, K. SHOME’, M.S. FINKEL*, C. LAGENAUR3 and N-F. ZAIDI’ Departments of Physiology’, Medicine-Pharmacologg, and Neuroanatomy and Cell Science3, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Ab!5tElCt - A jO6 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca*+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodinereceptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the fphyslological’ Ca2+ release channel, we prepared ryanodineaffinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodineaffinity chromatography were identified as the low molecular weight Ca*+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca*+ release channels. Surprisingly, ryanodineaffinity columns did not retain JFPs but purified 106 kD Ca*+ release channels which are a minor component (O.l-0.3?4 of SR proteins.

Despite extensive studies, the physiological mechanism responsible for Ca*’ release from skeletal sarcoplasmic reticulum (SR) remains unknown [l]. Perhaps the most important contribution to our understanding of SR Ca*+ release has been the use of ryanodine, as a2fharmacological tool known to interfere with Ca release. The Ca*+ release channel was identified by several laboratories as the high molecular weight 565 kD junctional ‘foot’ protein (JFP) through its high afIInity binding to ryanodine (Z-61. The ryanodine receptor compIex

was purified by tracking detergent-solubilized SR proteins in sucrose gradient centrifugation using [3H]-ryanodine [6]. Incorporation of JFPs in planar bilayers resulted in a cation permeable channel which was bIcxz+kedby micromolar ruthenium red, rnillimolar Mg and activated by micmmolar Ca*+ or ryanodine and millimolar ATP 16, 71. These criteria implied that the JFP is the physiological Ca*’ release channel since similar properties were observed for Ca*’ re1eas.e from heavy SR vesicles [8, 91 and ‘native’ Ca*+ release channels 635

636

incorporated by fusing SR vesicles with planar bilayers [lO-121. Sulfhydryl-gated106 kLICa2’ release channels

Another intriguing feature of SR Ca2’ release is its reversible activation and inhibition by sulthydryl oxidizing and reducing agents 1131. Sulfhy 1 dY’ reagents were found to act at the Ca2+-inducedCa release process, trigger Ca2+release and block r3HJryanodine binding to SR proteins [14-161. Sulfhydryl chemistry was used to isolate a 106 kD protein from skeletal SR which was immunologically distinct from Ca2’,Mg2’-ATPaseand JFPs [17]. Reconstitution of 106 kD proteins in planar bilayers revealed the presence of Ca2’ release channels with similar features as those described for ‘native’ Ca2’ release channels [17, 181. In addition to the wellestablished modulators of Ca2’ release, 106 kD channels were also activated and inhibited by sulfhydryl oxidizing and reducing reagents and were locked into a closed state by 2-10 nM ryanodine [17,18]. In this report, we linked ryanodine to Sepharose 6B beads to prepare functional ryanodine-affinity columns and thus purify ryanodine mceptor(s).

CELL CALCIUM

injection. Protease inhibitors, phenyhnethylsulfonyl fluoride (1 mM) or leupeptin (1 ug/ml) were added to all solutions used to isolate SR vesicles. After the last centrifugation step, the SR vesicles (5-10 mg/ml) were suspended in a medium containing 0.29 M sucrose, 3 mM NaN3. 10 mM imidazole HCl, pH 6.9 and stored in liquid nitrogen until use. Protein concentration was determined by the method of Lowry et al. [20]. Preparation of anti-106 kLI monoclonal antibody afinity columns

A mouse monoclonal antibody (mAb:lF4) was raised against non-denatured, biotin-avidin purified 106 kD protein (as described by Zaidi et al. [16]) according to the method of De St Groth and Scheidegger [21]. Hybridoma supematants wem screened using immunoblot assays 1221. The lF4 mAb did not cross-react with denatured 106 kD protein or other SR proteins on Western blots. Alternatively, lF4 mAb was linked to Affigel-10 beads (Bio-Rad) to prepare immuno-affinity columns and thus isolate antigenic 106 kD channel proteins. SR vesicles were solubilized with CHAPS detergent (1.25 mg SR/ml) in 20 ml of: 1.6% CHAPS, 1 M NaCl, 40 mM Tris, pH 7.0. The solubilized proteins were passed through the 3 ml mAb column then washed Materials and Methods with 60 ml of the same buffer. Antigen bound to the column was eluted with 0.1 M diethylamine, at Preparationof skeletaland cardiac SR pH 11.5 in 10 fractions (1 ml each). The washes and elutions of the immuno-affinity column were Skeletal SR vesicles were prepared from rabbit analyzed by silver stained SDS-PAGE, Western white skeletal muscle as previously described 1141. blots or immunodot blots. After the last centrifugation step, the vesicles were suspended at 10-12 mg proteinlml in a medium Preparationof ryanodine-a$initycolumns containing 0.9 M sucrose, 10 mM HEPES, pH 7.0 and stored in liquid nitrogen until use. In some Ryanodine (100 mg) was dissolved in 0.1 M experiments, junctional SR were prepared as prev- Na2HP04 (pH 12.0) and incubated with 1 g of iously described [8] in the presence of DIFP (1 mM) epoxy-activated Sepharose 6B (Pharmacia) at 23°C and EGTA (2 mM) to prevent proteolytic break- for 24 h. The beads were washed twice with 10 ml down of 565 kD feet proteins. of distilled water and remaining active epoxy groups Cardiac SR vesicles were prepared from canine on the beads were blocked by incubating with 1 M ventricular tissue as described by Chamberlain et al. ethanolamine, 0.1 M NazHPO4, pH 12.0 for 5 h. [19]. Hearts from mongrel dogs (used as donor The beads were then washed with 10 ml of 0.1 M animals in liver transplantation experiments) were acetate, 0.5 M NaCl at pH 4.0 followed by 10 ml of provided by the Department of Surgery, after the 0.1 M borate, 0.5 M NaCI, pH 8.0. The column was animals were sacrificed by lethal anaesthetic stored in 1 M NaCl, 20 mM NaPIPES, pH 7.2 at

RYANODINE-AFFINITYCHROMAT.TOPURIFYSRCaIONCHANNELS 4°C until use. The ryanodine concentration on the

columns was determined by using [3H]-ryanodine to track the amount of ryanodine washed away after each step. At the end of the procedure, 10 pl of beads were removed from the column, sonicated in scintillation fluid (10 ml) and counted to measure [3H]-ryanodine on the column. Columns were used an average of 12 +_ 2 times before losing their specificity. Purification of 106 kD ryanodine-afinity columns

channel

proleins

by

Solubilized SR proteins (25-50 mg) were incubated in a ryanodine-affinity column for 2 h with continuous shaking in a medium (10 ml) known to maximize ryanodine binding to its receptor [6]: 1 M NaCl, 20 mM NaPIPES, 1.6% CHAPS, 60 pM ionized Ca2+, 5 mM ATP, 0.25 mM PMSF, 1 pg/ml leupeptin, pH 7.2 at 4°C. Excess solution in the column was drained (run-through), the column washed 3 times (10 ml each) with a medium identical to the binding medium, for 30 min per wash, with continuous shaking before drainage, followed by 3 elutions with a medium known to dissociate ryanodine from its receptor (each elution was 3 ml lasting 15 min under continuous flow through the column). Run-&roughs, washes and elutions were analyzed by silver stained SDS-PAGE and immunoblots. Two elution media were extensively used (n = 24 each) to dissociate ryanodine-receptors bound to the ryanodine on the columns. Elution medium (A) consisted of: 1 M NaCI, 5 mM MgClz, 40 mM Trismaleate, 1.6% CHAPS at pH 7.2. Elution medium (B) consisted of: 10 mM NaCl, 40 mM Tris-maleate at pH 7.2. In the present experiments, the incubation of solubilized SR proteins in ryanodine columns, the washes and elution steps were all carried out at 4°C. Given the temperature dependence of ryanodine binding to SR, ryanodine-affinity columns were also run (i.e. incubation, washes and elutions) at higher temperatures. At 23” and 37°C. the incubation time of solubilized proteins in ryanodine columns could be reduced from 2 h to 90 and 30 min respectively. Higher temperatures did not alter the outcome of these experiments and ryanodine-columns still

637

purified 106 kD Ca2’ channel and did not retain 565 kD JFPs. However, increasing temperatures increased the amounts of ATPase sticking to the columns which contaminated the 106 kD eluted-off by high Mg2+ or low NaCl media. In 5 experiments with skeletal SR and 2 experiments with cardiac SR, phospholipids were added to the incubation wash, and elution media in attempts to improve: (i) the binding of 565 kD JFPs to ryanodine affinity columns; and (ii) the incorporation of ryanodine-affinity purified protein in planar bilayers. The phospholipid (3 mg/ml) composition in the solutions was the same as that used to form bilayers and consisted of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine at a ratio of 5:3:2. Cardiac SR vesicles were solubilized, incubated in ryanodine-affinity columns using the same protocols and reaction media as for skeletal SR. Competition of free (Rf) versus immobilized (Ri) ryanodine The competition of 13H]-ryanodine binding to SR protein by ryanodine immobilized on beads was measured using standard techniques. SR proteins (100 pg) were solubilized in 0.5 ml of 1 M NaCl, 20 mM NaPlPES, 1.6% CHAPS, 60 pM ionized Ca2’, 0.25 mM PMSF, 1 mg/ml leupeptin at pH 7.2 and incubated for 2 h with 10 nM of free (Rr) [3H]-ryanodine (60 Ci/mmol [9,21-3HJ-ryanodine, NEN, Wilmington, DE, USA) plus various volumes of Sepharose beads obtained from a ryanodineaffinity or ‘glucose’ column. The concentration of ryanodine immobilized in a volume of Sepharose beads (Ri) was determined by adding low levels of ryanodine isotope during the preparation of ryanodine columns. After a 2 h incubation, proteins were precipitated along with 1 ml of y-globulin (0.25% in 50 mM Tris buffer, pH 7.0) by adding 1 ml of polyethylene glycol (24% in 50 mM Tris buffer, pH 7.0). Proteins were filtered on Whatman filters (1.0 pm, presoaked in 1% polyethylenimine) using a filtration manifold. Filters were washed twice with 5 ml of polyethylene glycol (8% in 50 mM Tris buffer at pH 7.0), placed in 10 ml of scintillation fluid and counted in a Packard Scintillation Analyzer.

638

CELL CALCIUM

B

ANTI-ATPaw R

KD

-200

-92 -69

-46

-30

-21 -14

Fig. 1 Purification of 106 kD proteins by mAb affinity mhmms Solubilized

SR proteins (1.25 mg SR/ml in 20 ml of: 1.6% CHAPS, 1 M NaCl, 40 mM Tris, pH 7.0) wem passed through a mAb

affinity column (lane R) then washed with 60 ml of the same buffer (Wt to W3).

Antigen bound to the column was eluted with 0.1 M

diethylamine, at pH 11.5 in 10 fractions (1 ml each) to dissociate antigen from Ab (lanes l-10) (A): Fractions eluted of the column were analyzed by SDS-PAGE, followed by silver staining. the expected pattern of junctional SR proteins including JFPs and ma$r band of ATFax. after three washes with identical medium (60 mane). @lane).

Lane R: column run-through (8 11) has

Lanes W: proteins removed from the column

Lanes l-10: gmdual elution of antigenic proteins at about 100 kD at pH 11.5 (60

The lower two protein bands were identified as IgG fractions through a partial amineacid

sequence

(B): The same protein fractions were concentxated S-fold, analyzed on a Western blot [17], stained with anti-A’IPase mAb to detect Ca”,Mg”-ATPase.

Lane R: column run-thmugh, highly enriched with ATPase (60 pl/huz).

effective removal of ATPase from the column (60 PI/lane).

Lanes W: washes at pH 7.0 indicated

Lanes l-10: elutions of 106 kD proteins at pH 11.5 did not react with

anti-ATPase mAbs (60 PI/lane)

Reconstitution of ryanodine-a@@

pw@ed 106 k9

in planar bilayers

Ryanodine affinity purified 106 kD proteins (0.1-0.4 pg/ml) were incorporated in MuelIerRudin-type lipid bilayers consisting of phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine at a ratio of PE:PS:PC = 5:3:2 with a total lipid concentration of 50 rr@ml in decane. The bilayers were formed across an 80 or 150 pm diameter hole in a Kynar cup, as previously described Il81. An Axopatch 1D (Axon I.nstmmems. Foster City, CA, USA) amplifier with a CV-3B headstage was used to measure picoampere current fluctuations. Data were digitized (Instrutech Corp.,

Model VR-10) and stored on video-tape for subsequent analysis. Analog data output from the video recorder to the Instrutech were digitized with an analog-todigital converter (Labmaster TM-40, Scientific Solutions, Solon, OH, USA), transferred to computer memory (IBM AT/386) and analyzed using pClamp software (Axon Instruments). The Na+ conductance gNa+ of the 106 kD Ca2+ release channel was measured after adding protein to the &-side of the chamber containing: 20 mM NaPIPES, 20 J&I CaCb, 20 pIvI Na2EGTA pH 7.4 and either 500 or 250 mM NaCl. The CHAPS concentration was kept below 64 pg/rr$ that is, far less than the concentration that affected the conductance of bilayers, in the absence of protein.

RYANODINE-AFFINITY

CHROMAT.

TO PURIFY SR Ca ION CHANNELS

The truns-side of the chamber was held at virtual ground and contained the same NaCl solution (i.e. symmetrical NaCl solutions).

Materials

The anti-Ca2+,Mg2+-ATPase monoclonal antibody was the generous gift of Dr David MacLennan. All reagents were of analytical grade. CHAPS was purPIPES, chased from Boehringer-Mannheim. I-IEPES, Tris, ATP, EGTA, SDS-PAGE and molecular weight standards were purchased from Sigma. Affigel-10 beads were purchased from Bio- Rad; goat anti-mouse IgG linked horseradish per- oxidase was purchased from Organon Teknika Cappel (Malvern, PA, USA). Ryanodine was pur- chased from AgriSystems International (Windgap, PA, USA) and radiolabelled [3H]-ryanodine from New England Nuclear (Wilmington, DE, USA). Lipids were purchased from Avanti Polar Lipids Inc., (Birmingham, AL, USA).

Results Figure 1A shows a purification of 106 kD Ca2+ channel proteins by monoclonal antibody (mAb)affinity chromatography followed by an SDS-PAGE analysis, stained with silver. Solubilized junctional SR proteins were incubated in the column at pH 7 to bind antigen to mAb. unbound proteins were ‘runthrough’ (lane R), the column was washed 3 times (lanes W) then antigens were eluted by passing buffer at pH 11.5 to dissociate antigens from the column (lanes l-10). The antibody- affinity column purified a 106 kD protein band (lower bands consisted of IgGs leakin from the column) which Z!+ did not contain Ca2+,Mg -ATPase, as verified by a Western blot stained with an anti-ATPase mAb (Fig. 1B). Note the lack of higher molecular weight proteins above 106 kD bands in lanes 2-8 (Fig. 1A). The protein isolated in Figure 1 using a monoclonal antibody (lF4) cross-reacted with the polyclonal antibod raised against biotin-avidin purified 106 I+ kD Ca release channel which was previously described by Hilkert et al. [181.

639

Ryanodine-ajjkity chromatography We used ryanodine as an affinity probe linked to an immobilized matrix in attempts to identify high and low affinity ryanodine binding sites [23-261. Ryanodine was linked to epoxy-activated Sepharose 6B via one of its 8 hydroxyl groups. The chemical reaction is equivalent to the alkylation of the ryanodine molecule by the oxirane ring on epoxyThe groups most activated Sepharose 6B. susceptible to alkylation by the oxirane moiety are the pyrolle NH, and hydroxyl groups at site 15, then hydroxyl groups 4 or 6; hydroxyl group 10 is expected to be less reactive ([27], IN. Pessah, personal communication). Since any one of ryanodine’s hydroxyls can be anchored to Sepharose-6B, an unknown fraction of these ryanodine molecules will no longer bind to the receptor because the binding region on the ryanodine molecule is modified or obstructed by the long spacer arm anchoring ryanodine to Sepharose beads. To determine the total concentration of ryanodine linked to the column, [3H]-ryanodine (100 mg/3 ml of Sepharose 6B) was incubated with epoxyactivated Sepharose beads and the tritium label was monitored during each step of the procedure. The difference of tritium label in the initial incubation medium minus the losses of label at every step was equal to the amounts of label bound in an aliquot of Sepharose beads. The 13H]-ryanodine bound to Sepharose columns (3 ml) was in the range of 230-380 @I with an average of 286 I.&I (n = 14). Even if 1% of the ryanodine in the column retained its native binding properties, the concentration of functionally active Iyanodine would still be significantly greater than the association constant of ryanodine to its high (KD = l-5 nM) and low (KD = l-2 pM) affinity binding sites. Thus, ryanodineaffinity columns should purify ryanodine receptor(s) and SR Ca2+ release channels. In Figure 2, ryanodine columns were incubated in a medium known to maximize ryanodine binding to CHAPS solubilized SR proteins (see Materials and Methods). After 3 washes, ryanodine receptors were eluted by washing the columns with media known to cause the dissociation of receptor(s). In Figure 2A, ryanodine-receptors bound to the column were eluted with elution medium (A) in which Ca’+

640

CELL CALCIUM

A

B

116 97 66 205

116 97 66

F

E2

anti-S-106 (lF4)

mAb

anti-ATPnse

mAb

?%

anti-SG-106 (lF4)

mAb

anti-ATPase

mAb

Amid+black Amido-black

WI

w3

El

E2

E3

E3

),

,*

“” ;

U

Controls

Fig. 2 Ryanodine afllnity columns purify 106 kD proteins Ryanodine was linked to epoxy-activated Sepharose 6B to form a 3 ml ryanodine column containing 245 pM ryanodinc. Solubilized SR proteins (30 mg) were incubated in the column for 2 h with wntinuous shaking, in a medium (IO ml) known to maximize tyanodine binding to its receptor [6]. Excess solution in the column was drained (run-through), the column washed 3-times (each wash was an incubation with identical binding medium (10 ml) for 30 min with shaking before drainage); and three elutions in a medium that dissociates ryanodine ftom its receptor (each elution, 3 ml of dissociation medium lasting 15 min under continuous flow). Run-through (15 pI out of 3 ml), three washes (52 pI out of 10 ml) and three elutions (52 pl out of 3 ml) were analyzed by SDS-PAGE followed by silver staining (Ieft). These samples were spotted (5 pl each) on nitrocellulose and probed with (i) IF4 anti-106 kD mAb (ii) anti-ATPase mAb, (iii) stained with amidoblack to compare total protein in each fraction, and (iv) contmls lacking a primary antibody (A) (Left above)Lane 1 : run-throughcontaineddominant bands of ATPase and high molecular weight JFPs. Lanes 2-4 : three washes contained protein bands primarily at 100 kd. Lanes 5-7 : ptoteins dissociated fmm the column by passing a medium, with 5 mM MgCl2 nut lacking Ca’+ and ATP. The dominant protein band in tbe three elutions was at 100 kD with no high molecular weight proteins. Inununoblots indicated that proteins eluted with high Mg2+ and low Ca2’ reacted with anti-106 kD mAbs and not antiCa2+,M$-ATPase mAbs (B) (Right above) Lane 1 : Run-through with a typical pattem for SR protein. Lanes 2-4: washes from the column. Lanes 5-7 : proteins dissociated from tbe column by reducing NaCl from 1M to 10 mM. lnuntmoblots indicated again that the dominant protein eluted fmm the ryanodine column was 106 kD protein (C) (L&-hand column on facing page) Control : The association and dissociation of 106 kD proteins from tbe column was dcpcndent on immobilized tyanodine on the column. SR vesicles were pm-incubated for 2 h with 10 nM ryanodine before solubilization with 1.6% CHAPS then passed through the tyanodine cololumnas described for (A). Lane 1 : Run-through revealed pattern of SR proteins. Lams 2-4 : washes with binding medium. Lanes 5-7 : elution of tyanodine receptor as for (A) by deleting ATP and Ca2+ and raising Mg2+ (5 mM). Innnunoblots indicated that soluble ryanodine (10 nM) competed with tyanodine bound to tha column and blocked the column’s retention of 106 kD proteins

was reduced from 100 to 2 @.4. ATP (5 mM) was deleted and free Mg2’ was raised from 0 to 5 mM. In Figure 2B elution medium (B) was used to

collect xyanodiae-receptors from the column. In this case, NaCl was reduced from 1 M to 10 m.M, ATP and Ca2’ were deleted. In both cases, ryanodine-

RY ANODINE-AFFINITY

CHROMAT.

TO PURIFY SR Ca ION CHANNELS

641

Four types of controls indicated that ryanodine columns isolated 106 kD proteins because some of the immobilized-ryanodine (Ri) retained tire binding characteristics of ‘native’ or free ryanodine (Rr).

C

E.l

Fig. 2C See opposite page for caption

affinity columns purified a protein band at about 100 kD and did not retain high molecular weight JFPS. Immunodot blots using anti-106 kD and anti-ATPase monoclonal antibodies indicated that these columns (n = 14) consistently (12 experiments per column purified a protein identified as the a 106 kD Ca + release channel and not Ca2+,Mg2+ATPase. The anti-106 kd mAb (lF4) interacted with non-denatured proteins and thus could be used to detect 106 kD proteins on immunoblots but not on Western blots. The mAb-affinity columns prepared with lF4 mAb demonstrated the selectivity of its immune reaction by extracting 106 kD Ca2’ release channels from solubilized SR proteins (Fig. 1). The presence of lipids (n = 5) in the incubation and elution media did not significantly increase the retention of 106 kD proteins by ryanodine-affinity columns nor did the columns extract 565 kD JFPs. The addition of lipids was primarily used to improve the probability of channel incorporation in planar bilayers.

(1) An excess of Rr was added to solubilized SR proteins during the incubation of the reaction mixture in the column to determine if Rf competed with the binding of 106 kD proteins to the columns. As shown in Figure 2C, Rf (10 nM) competed with Ri such that the columns no longer purified 106 kD or any other SR protein. This outcome was reproduced with 10 nM (n = 3) and 100 nM (n = 3) Rf in the incubation medium. (2) Conversely, we tested for the ability of Ri on beads to inhibit [3H]-Rf binding to solubilized SR proteins. [3H]-Rr binding to SR proteins was measured in the presence of various concentrations of Ri by adding known volumes of Sepharose beads (O-105 p.l). As shown in Figure 3, Ri competed and inhibited [3H]-Rr binding to three different preparations of SR proteins (open circles, filled circles, open circles with central dot). On the other hand, control beads (glucose linked to Sepharose 6B) did not alter [3H]-Rr binding (inverted open triangles). Ri competed with the binding of Rr to SR proteins with an Ic50 of 1 p.M and under the present binding conditions with [Rf] = 10.0 nM; Kr(Ri) = 500 nM instead of 3-5 nM for Kr(Rr). The results indicate that either the KD of Ri was reduced by a factor of about 100 or 1% of Ri (2-4 pM) remained active and capable of binding to SR proteins. The ryanodine linked to the beads (Ri) was taken from a ryanodine-affinity column that was used to purify functional 106 kD Ca2+ release channels and had been subjected to the full treatment that we developed for the preparation of ryanodine-affinity columns. (3) To determine if 106 kD proteins bind to Sepharose 6B in a non-specific manner, the above experiments were repeated using ‘sham’ columns substituting glucose for ryanodine. SR proteins were detergent solubilized and passed through ‘glucose’-affinity columns as described for

CELLCALCIUM

642

ryanodine-affinity columns. Four sham columns were prepared and tested twice each; in all cases such ‘glucose columns’ did not retain SR proteins indicating that the purification of 106 kD proteins was dependent on the linkage of ryanodine to the Sepharose beads (data not shown). (4) Exposure of [3H]-ryanodine to Na2HP04 buffer at pH 12.0 for 24 h at 23°C (but without linking ryanodine to the beads) did not alter binding activity (i.e. cpms were 90 to 96% of control [3H]-ryanodine binding) to solubilized SR proteins. Thus, the extraction of 106 kD proteins by ryanodine columns was dependent on its interaction with active ryanodine linked to the beads. CHARS solubilized SR were incubated in a ryanodine affinity column in the presence of phospholipids (see Materials and Methods). The column was washed and ryanodine receptors were eluted off the column with elution medium (A) which also contained phospholipids. Ryanodineaffinity purified 106 kD proteins were reconstituted in planar bilayers and thus revealed the presence of functional SR Ca2’ release channels in planar bilayers. As shown in Figure 4, single channel fluctuations were measured in symmetrical NaCl (0.5 M) solutions (Fig. 4a). An addition of ryanodine (10 nM) to the franr-side had no effect on channel activity (Fig. 4b,c). After 15 min of uninterrupted recordings, an addition of ryanodine (10 nM) to the &side (Fig. 4d) resulted in closure of the channel within 30 s (Fig. 4e,f). The molecular entity responsible for these single channel fluctuations was not likely to be contaminating levels of 565 kD JFPs because high molecular weight proteins were not detected in silver-stained gels. Moreover, low concentrations (nM) of ryanodine were shown to have no effect on purified 565 kD proteins incorporated in planar bilayers [6, 71. Thus, if 565 kD JFPs were incorporated in these bilayers instead of 106 kD and JFF% were responsible for the channel fluctuations, then nM concentrations of ryanodine should have had no effect on channel activity. On the other hand, the incorporation of immuno-affinity purified 106 kD

revealed the presence of Ca2’ release channels that were sensitive to sulthydryl reagents and could be locked in a full closed state by 2-10 nM ryanodine. Ryanodine-aj+inity columnsput@ a cardiac 106 kD likeprotein Ryanodine-affinity columns were used to isolate a low molecular weight or 106 kD like Ca2’ release Volume of Beads (pl) 0

20

40

60

80

100

120

100

60

20

yanodine-Beads

b\

0 0

4

8

12

16

20

24

28

[Ryanodine] in Beads (PM)

Fig. 3

Immobilized ryawdine (RI) competes with the binding of

free [3H]-ryanodine

(Rr) to SR proteins

Free [3H]-ryanodine the presence Sepharose affinity

Rr biiding

of various

6B beads I1 [31]. cohunns

were

to SR proteins

concentrations

Ri from three different

incubated

with

proteins (100 pg) from three separate nM 13H]-Rr. inhibited circles.

open circles

standard

error

consisting

was

mean

with central within

alter

ryanodine

of

prepamtions;

three

experiments

SR

linked to beads, Ri filled

For each data point the

10% of its value.

of bead concentration.

binding

Control Sepharose

beads 6B did

to SR proteins

Each data point represented done

the error bars represented

mean values (inverted open uiangles).

to

plus 10

of [Rt] (open circles, dot).

in

tyanodine-

CHAPS-s&bilked

of glucose linked to epoxy-activated

not signiticantly function

as a function

linked

vesicle preparations

In all three batches of ryanodine

Rr binding

was measured

of ryanodine

with

three

the standard

separate

as a the SR

etrors from

Thus beads alone did not

interfere with ryanodine

binding and qanodine linked to the beads

inhibited [3H]-ryanodine

binding to SR proteins

RY ANODINE-AFFINITY

CHROMAT.

TO PURIFY SR Ca ION CHANNBLS

40 mv

d.

‘0

i-1 0 nV

tJ.P.

Rynnodine,

cis-side

10 &

Fig.

4

Ryanodine-affinity

release channels

purified

106 kD proteins

Protein

(10 ul from Fig. 2B, lane 5) was incorporated

c&side

of a planar bilayer in symmetrical

mM NaPIPES,

20 pM CaClz and 20 pM EGTA, pH 7.4, at 23’C described

(A) Incorporation

of the 106 kD protein produces

apparatus

with a Na+ conductance,

[ 17, 18 ] gt&

(B) An addition

of 10 nM ryanodine

effect

channel

on single

ryanodine

from the

NaCl (0.5 M NaCl. 20

using a previously fluctuations

am Ca*+

and are blocked by 10 nM ryanodine

activity

(10 &I) from the c&side

single channel

- 359.5 pS to the truns-side

but a subsequent

had no

addition

of

resulted in complete channel

closure within 60 s

channel from cardiac SR since sulfhydryl reagents (i.e. heavy metals and reactive disultides) were also found to induce Ca2’ release from cardiac SR vesicles [28, 291. A new ryanodine-affinity column

643

that had never been exposed to skeletal SR proteins was prepared to isolate cardiac ryanodine-receptors. Ryanodine-affinity columns were assigned for studies of either cardiac or skeletal SR but were never exposed to both types of SR to avoid crosscontamination of proteins. Cardiac SR was solubilized and passed through ryanodine-affinity columns using the same conditions as described for skeletal SR (Fig. 2), except that phospholipids (3 mg/ml, PE:PS:PC = 5:3:2) were added to all solutions. An analysis of such an experiment using silver stained SDS-PAGE and immunodot blots is shown in Figure 5 (A & B). Cardiac SR proteins were incubated for 2 h with ryanodine linked to the column, under conditions that maximize ryanodine binding to cardiac SR. The column was drained and the flow-through contained the bulk of cardiac SR proteins (Fig. 5A, F, lane 1). The subsequent 3 washes contained protein bands at about 100 kD (Fig. 5A, W, lanes 2-4). This was followed by two elutions in a medium containing 5 mM MgCl2 and no added Ca2’ which contained protein bands (Fig. 5A, E. lanes 5-6) migrating above 100 kD or just above the dominant band of Ca2+,Mg2+-ATPase seen in the flow-through (Fig. 5A, F, lane 1). Immunodot blots of the flow-through (Fig. 5A, lane 1) washes 1 and 3 (Fig.SA, from lanes 2 and 4) and the two elutions (Fig.SA, lanes 5.6) are shown in Figure 5B). The dot blots indicated that the 106 kD protein was detected weakly in wash 1 and strongly in wash 3 and elutions 1 and 2 (Fig. 5B, top immunohlots). Furthermore, anti-ATPase immunoblots indicated a strong immune reaction to ATPase in the flow-through and wash 1, did not detect the presence of ATPase in wash 3 and both elutions (Fig. 5B, 2nd row of dot blots). As shown in Figure 6, (top traces) the incorporation of ryanodineaffinity purified 106 kD protein (from Fig. 5, lane 5) in planar bilayers revealed the presence of a Ca2+ channel. In symmetrical Na+ solutions, the channel exhibited a prominent full-open state with a sodium conductance of 385 pS and 2 subconductance states of 197 and 113 pS or approximately l/2 and l/4 of the full-open state. Ryanodine-affinity chromatography purified a protein from canine cardiac SR which cross-reacted with a monoclonal antibody (lF4) raised against the rabbit skeletal 106 kD (n = 6).

644

CELL CALCIUM

A

M.W.

F W W

kDa

1

2 3

W

E E

Fig. 5 Ryamdine-affinity puritication of cardiac 106 kD protein Caniae cardiac SR proteins (50 mg) were solubilized in CHAPS as described in Materials and Methods, incubated for 2 h at CC with continuous shakiog in a ryanodine-at%@ column containing 345 ph4 of linked ryamdine. The column was drained to collect the flow tbrougb, washed and drained witb an identical medium 3-times, tbcn the proteins specifically linked to the immobilized ryanodine were eluted twice by passing a medium with 5 mM MgCk and 200 pM EGTA to dissociate ryanodine from its binding site(s) (A) Silver stained SDS-PAGE analysis of canine cardiac SR through a ryatmdine-affinity column. Lane 1, E 15 pl of flow through, lanes 2-4, W: 65 pl of sequential washes 1-3 and lanes 5,6, E: 65 pl of elutions 1 and 2. The mlumn selectively retained a protein band at about 100 kD (B) Immunodot blots from the column indicate that a cardiac protein cross-reacts with the monoclonal antibody raised against the skeletal 106 kD protein. The cardiac 106 kD protein appeared in washes l-3 and elutions 1 and 2 whereas ATPase appearedin the flow through (F) and wash 1. Amidoblack staining indicated the relative distributionof protein in the various fractions and the controls (lower blots) demonstrated the specificity of these reactions

4 5 6

2Q5

116 97

66

F anti-SG-106

WI

w3

El

E2

mAb ,---. ,,

anti_ATpase

mAb

.” *_, ‘!

Amido-black Control

Discussion

complex. JFPs were not retained by these Columbus but were present in the run-through and washes

The data show that yyodine affinity columns isolated 106 kD SR Ca release channels but not 565 kD junctional feet proteins which are thought to form tetramers that comprise the ryanodine-receptor

from the columns. These results are surprising because the conditions used to associate and dissociate SR proteins to the columns were identical to the conditions reported for the binding and unbinding of

RY ANODINE-AFFINITY

CHROhIAT.

Ryanodine-Affinity 106-kDa o.

NaCl

TO PURIFY SR Ca ION CHANNELS

Purified

Ca*+ Release mM),

(250

+30

mV

Cardiac Channel

H.P.

30 20 10 i

OL 5s

PA

b. Fast

30

Sweep

(4

s)

20 10 0

0.5 s

PA

Wg. 6 Reconstitution

9 No’ (PSI

--

of cardiac

0 F.O.

385.0

l/2

197.0

l/4

113.6

106 kD in planar bilayers

(A) Protein from lane 5 of Figure SA was incorporated

in a planar

bilayer in symmetrical NaCl (250 mM) and revealed the presence of a cationic

channel

blocked by ruthenium

that was activated

(B) A current to voltage relationship NaCl

the cardiac

pS for its full-open

by ATP (3 r&f)

and

red (5 @I) (not shown)

cationic

channel

indicated

that in symmetrical

had a Na+ conductance

state and two subconductance

of 385

states of 197 and

113.6~s

JFPs to [3Hl-ryanodine. The ryanodine-affinity purified 106 kD protein can be incorporated in planar bilayers to produce functional Ca2’ release channels. The 106 kD channels were blocked by nanomolar ryanodine from the cis (but not rrunr) side which corresponded to the side of the channel that interacts with ATP, Mg2+, Ca2+, ruthenium red

645

on the cytosolic side of the channel. The ryanodineafftity columns also purified a 106 kD like channel protein from canine cardiac SR which was immunoreactive with a monoclonal antibody against the channel from skeletal muscle. Controls indicated that a significant fraction of the covalently-linked ryanodine retained the binding characteristics of native ryanodine. The pharmacological agents that enhance or inhibit ryanodine binding to its receptor were also found to enhance or inhibit the binding of 106 kD proteins to ryanodine-affmity columns. Moreover, the purification of 106 kD proteins by ryanodine columns was independent of the procedure used to link ryanodine to a matrix. We also treated ryanodine with cyanogen bromide to form cyanate ester groups at one of its hydroxyl sites. The cyanate ester groups were then linked to diethylaminoethyl Sephadex (DEAE) to immobilize ryanodine on a DEAE column which also purified 106 kD proteins and not JFFs (not shown). Thus it is difficult to argue that the association and dissociation of functional 106 kD channels (rather than JFFs) as a function of [NaCl], ATP, [Ca2’lfr~ and [Mg2+lfree is a coincidence caused by a ‘fortuitous’ chemical alteration of ryanodine during its linkage to Sepharose 6B. The study contradicts a large body of evidence identifying tetramers of 565 kD JFPs as the Ca2+ ryanodine receptor complex and the physiological SR Ca2+ release channel. One possible resolution would be that 106 kD proteins are proteolytic fragments of 565 kD JFPs which include the pore of the Ca2’ release channel and its gating machinery. Another possibility is that 106 kD proteins are the product of post-translational modifications of JFPs. However, these hypotheses are not consistent with the present data which show that ryanodine linked to beads can compete with [3Hl-ryanodine binding to SR yet do not extract 565 kD JFPs. These hypotheses are also in conflict with observations that sulfhydryl reagents trigger SR Ca2’ release from vesicles and skinned fibers and activate single channels in biiayers [13-16, 18, 28-301; yet these reagents did not tag 565 kD JFPs [17]. Altematively, the resolution may lie in alternative interpretations of ryanodine binding studies with JFPs in that 106 kD proteins have most likely been co-purified with JYPs but have been overlooked.

646

(1)

CELL CALCIUM

The identification of JFPs as the ryanodine receptor has been primarily based on the co-migration of [3H]-ryanodine and JPPs in linear sucrose gradients [2, 6, 73. However, other protein bands (including 106 kD channels) co-migrate with 565 kD JPPs and [3H]-ryanodine in sucrose gradients [171 and proteins contaminating JPPs could be ryanodine receptors and Ca2’ release channels that might be incorporated in planar bilayer experiments. Moreover, an attempt at further purification of JPPs by removing 100 kD proteins co-migrating along with JPPs in sucrose gradients, resulted in a gradual reduction and elimination of ryanodine binding to purified JFPs 131I.

(2) The stoichiometry of high-affinity tyanodine binding to JFPs is 1 to 4; that is, one ryanodine binds to 2.26 MD protein. On the other hand, if ryanodine binds to 106 kD proteins with a 1 to 1 ratio, then a 6-S% contamination of 106 kD proteins could account for channel activity attributed to JFPs. (3)

Perhaps the strongest evidence in support of JPPs as ryanodine receptors is the cloning of the cDNA coding for 5037 amino acid residues comprising the JFPs and their expression in Chinese Hamster Ovary (CHO) cells [32]. The expression of JFPs in CHO cells was demonstrated by detecting [3H]-ryanodine binding (4 pmol/mg protein) in cells that do not normally bind ryanodine [32] and by eliciting Ca2’ release from internal stores with caffeine (l-50 mM) or ryanodine (100 p.M), with the same outcome whether ryanodine was added extracellularly or injected in the cytosol [33]. However, the lack of binding and modulation of Ca2’ release by ryanodine in untransfected CHO cells may be a reflection of the sulthydryl oxidation-reduction state of cryptic receptors rather than lack of In support of this hypothesis, receptors. lowering the redox state (i.e. by adding a sulfhydryl reagent) in CHO cells and mammalian oocytes was found to cause Ca2’ release from internal stores 134, 351. In addition, ryanodine was found to inhibit intracellular Ca2’ oscillations elicited by inositol( 1,4,5)tris-

phosphate in hamster oocytes (Dr Karl Swarm. personal commnnication). The activation of hamster oocytes by sulthydryl oxidizing agents indicated the presence of sulfhydryl-sensitive Ca2’ release channels [35]. Ultimately, the sequence analysis of the 106 kD protein and expression of cloned 106 kD protein gene will be necessary to determine the relationship between the 106 kD and JF’Ps. Regardless of the outcome, this lower molecular weight protein which retains the essential pharmacological gating properties of Ca2+ release channels, may prove to be a more tractable molecule for studies of channel structure-function relationship.

Acknowledgements Thanks are due to Jodi Segal, Christina Teramana and Patricia

Will for their technical assistance, William Hughes, our departmental machinist, for the construction of Kynar cups and Alice Diven for typing the manuscript. This work was supported by grants from the National Science Foundation DCB-8918672. the Western Pennsylvania Affiliate of the American Heart Association and a James Shannon Award from the National Institutes of Health, R55 AR40836 to G. Mama.

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RYANODINE-AFFINITY

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647

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