493BELTRÁN ET AL. Biol Res 39, 2006, 493-503Biol Res 39: 493-503, 2006 BRFast kinetics of calcium dissociation from calsequestrin

MARIANELA BELTRÁN*, GENARO BARRIENTOS and CECILIA HIDALGO

Instituto de Ciencias Biomédicas and Centro FONDAP de Estudios Moleculares de la Célula,Facultad de Medicina, Universidad de Chile, Santiago, Chile.* Present address: Laboratorio Andrómaco S.A., Santiago, Chile

ABSTRACT

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolatedjunctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium withcalcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsicfluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25ºCcalcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s-1) thanin solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting duringexcitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease forcalsequestrin in solution or forming part of junctional membranes was significantly slower than the rates ofcalcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrinassociated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s-1) than the slower component (k = 6.9 s-1), which had the same k value as forcalsequestrin in solution. These combined results suggest that the presence of calsequestrin at highconcentrations in a restricted space, such as when bound to the junctional membrane, accelerates calciumdissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.

Key terms: calcium-binding proteins, ryanodine receptors, sarcoplasmic reticulum, calcium release kinetics,excitation-contraction coupling, skeletal and cardiac muscle.

Corresponding author: Prof. Cecilia Hidalgo, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad deChile, Casilla 70005, Santiago 7, Chile, Tel.: (56-2) 978 6510, Fax: (56-2) 777 6916, Email: chidalgo@med.uchile.cl

Received: September 24, 2005. Accepted: October 26, 2005

PROLOGUE

On how I met Guayo and developed alife-long interest in calcium

I (CH) can still remember vividly the periodwhen, under Guayo’s guidance, I firstworked in calcium during the summer of1965. I had just obtained my professionaldegree as a Biochemist, and Mitzy Canessa,my biochemistry thesis advisor, had invitedme to work in Montemar with Guayo, whohad come for the summer from the US toresume his work on squid axons. He wastotally immersed in setting up from scratchan electrophysiological lab with theequipment he had brought from NIH, adaunting task considering the rather shorttime he had.

As a research project, he asked me tostudy calcium efflux from the squid giant

axon. We found that decreasing temperaturemarkedly reduced calcium efflux (Rojasand Hidalgo, 1968). This behaviorsuggested that an active plasma membranecalcium pump had a significant role oncalcium efflux. I had gathered experienceon the effects of mitochondrial inhibitors onactive sodium transport in the amphibianurinary bladder, so it was almost natural forme to investigate how mitochondrialinhibitors affected calcium efflux from theaxon. Surprisingly at the time, we alsofound that inhibition of mitochondriaincreased calcium efflux (Rojas andHidalgo, 1988). We proposed that thisincrease was due to inhibition of activecalcium accumulation into an intra-axonalcompartment, which in retrospect mighthave been the mitochondria.

Unfortunately, the summer and the squidsupply ended before I had time to

BELTRÁN ET AL. Biol Res 39, 2006, 493-503494

investigate whether external sodiumremoval affected calcium efflux, additionalexperiments envisioned by Guayo. So wemissed the opportunity to discover thesodium-calcium exchanger of squid giantaxons, which was reported in 1969 by thelate Peter Baker and coworkers.

It was not easy for me to work withGuayo. Although later we became verygood friends, he was under a lot of pressureat that time, a pressure that I felt acutely.Furthermore, I had decided to work onsquid axons mostly by the brilliant lecturesMario Luxoro gave on this subject in thebiophysics course that he taught tobiochemistry students, but Guayo had notime then for theoretical discussions. Hewanted results fast, so his interactions withme focused on the practical aspects of howto perform the experiments, and I was notvery happy with this approach.

I returned to Montemar on the summersof the years 1967 to 1969 for my Ph.D.thesis work, but during this period I did notwork again with Guayo or with calcium. Yet,he was a key person in helping me obtain apostdoctoral position at NIH in 1969, rightafter I had obtained my doctoral degree. Iwill always thank him for his extremelygenerous help during my doctoral thesis andlater at NIH, both in personal as well asscientific aspects. Through him, I developeda keen interest in calcium, an interest I keepvery much alive to this day.

For this reason, I decided to write anarticle in this issue in homage to Guayo onthe rate of calcium dissociation fromcalsequestrin, a key luminal protein of thesarcoplasmic reticulum that not only allowsthis organelle to store significant amountsof calcium but that also seems to regulatethe function of its calcium release channels.

INTRODUCTION

Calsequestrin (CSQ) – the most abundantluminal protein of skeletal and cardiacmuscle sarcoplasmic reticulum (SR) – bindscalcium with high capacity (40-50 moles permol) but relatively low affinity (MacLennanand Wong, 1971; Ikemoto et al., 1972;Cozens and Reithmeier, 1984; see Beard et

al., 2004, for a recent comprehensivereview). These properties allow CSQ to actas an effective intra-SR Ca2+ buffer, so thatwhen Ca2+ is transported actively back intothe SR, as required for muscle relaxation,CSQ binds Ca2+ and lowers the free [Ca2+]inside the SR lumen to levels ≤ 1 mM. As aconsequence, the SR can store large amountsof Ca2+, which can reach total concentrationsof up to 20 mM (Beard et al., 2004).

Calsequestrin is a highly acidic proteinwith approximately 50 Ca2+-binding sites permolecule. At neutral pH, monomeric CSQ insolution undergoes significant structuralchanges upon Ca2+ binding (Ikemoto et al.,1972). Thus, increasing [Ca2+] in the sub-mM range produces a conspicuous increasein CSQ intrinsic fluorescence and induces ashift in CSQ structure from a random coil toa structure containing significant a-helicalcontent (Ikemoto et al., 1972). Likewise, inCa2+-free conditions, decreasing solution pHfrom 8.0 to 6.0 induces significant structuralchanges, as reflected in marked increases ofCSQ intrinsic fluorescence; moreover, CSQintrinsic fluorescence does not increasefurther following Ca2+ addition at pH 6.0(Hidalgo et al., 1996). These results suggestthat Ca2+ and protons can both bindindependently to CSQ and induce the samechanges in CSQ structure that underlie theintrinsic fluorescence increase. Furtherincreasing [Ca2+] to levels ≥ 1 mM causesCSQ aggregation and polymerization (He etal., 1993).

In SR vesicles, CSQ forms a wide rangeof high molecular mass clusters (Maguire etal., 1997) and is attached to two junctionalface membrane proteins, triadin, andjunctin; CSQ forms a quaternary complexwith the RyR calcium release channelspresumably through its association withthese two proteins (Guo and Campbell,1995; Zhang et al., 1997), albeit directinteractions between CSQ and RyR alsomay take place (Murray and Ohlendieck,1998). It has been proposed that thestabilization of the quaternary SR proteincomplex composed by CSQ, junctin, triadinand RyR requires Ca2+ (Wang et al., 1998);yet increasing Ca2+ beyond 10 mM inhibitsCSQ binding to junctin and triadin, in vitro(Zhang et al., 1997; Wang et al., 1998).

495BELTRÁN ET AL. Biol Res 39, 2006, 493-503

Several reports indicate that, in additionto its role as luminal SR Ca2+ buffer, CSQalso regulates the activity of RyR Ca2+

release channels of skeletal and cardiacmuscle (see Beard et al., 2004). Increasingluminal Ca2+ in a range that promotes Ca2+

binding to CSQ stimulates caffeine-inducedCa2+ release kinetics from skeletal SRvesicles (Ikemoto et al., 1989). Likewise,increasing luminal [Ca2+] to 1 mM orincreasing luminal [H+] to pH 5.5 stimulatesthe kinetics of Ca2+ release from skeletal SRvesicles induced by Ca2+ and ATP (Donosoet al., 1995; Donoso et al., 1996). Thus,conditions leading to an increase in CSQintrinsic fluorescence and α-helical content,such as increasing luminal-free [Ca2+] to 1mM, stimulate the activity of RyR formingpart of skeletal muscle SR vesicles, whereasconditions that cause calsequestrin tounravel and adopt the random coilconfiguration would cause RyR channelinhibitions from the luminal side (Hidalgo etal., 1996). Yet, when using 1 mM free [Ca2+]in the trans (luminal) compartment, thesingle-channel activity of native RyR –measured in conditions that presumablyallow RyR incorporation into lipid bilayerswith CSQ, triadin, and junctin – ismaximally inhibited by CSQ. In contrast,under the same conditions, the purifiedskeletal RyR is not inhibited (Beard et al.,2002). These results have been interpreted asevidence of RyR inhibition by CSQ, whichwould be attached to the RyR channels inbilayers in the presence of 1 mM luminal-free [Ca2+] (Beard et al., 2002). Since in 1mM Ca2+ CSQ is expected to have asignificant α-helical content, CSQ seems toaffect differently native RyR when formingpart of isolated SR vesicles than whenincorporated in bilayers. A possibleexplanation for this different behavior mightbe that the high molecular mass CSQclusters found in the SR lumen (Maguire etal., 1997), which might be responsible forRyR activation in 1 mM [Ca2+], do notpersist in bilayer experiments.

Results obtained in skinned skeletalmuscle cells, which show that Ca2+ releaserates depend on Ca2+ loading (Lamb et al.,2001), also support a role of CSQ inregulating RyR activity. In intact

cardiomyocytes, increased or decreasedcardiac CSQ expression is an importantdeterminant of the Ca2+ storage capacity ofthe SR (Gyorke et al., 2004). It is noteworthythat the amplitude of depolarization-inducedCa2+ transients is significantly increased incardiac myocytes over-expressing CSQ andradically reduced in myocytes with decreasedCSQ expression. Furthermore, the duration ofactive Ca2+ release underlying these signalswas prolonged in cells over-expressing CSQand shortened in myocytes with decreasedCSQ expression (Gyorke et al., 2004). Theseresults strongly suggest that CSQ is anessential determinant of the Ca2+ releasingfunction of cardiac SR, since by influencingthe duration of the release process, CSQseems to control the amount of Ca2+ releasedto the cytoplasm.

The above results in intact cells indicatethat CSQ appears to function as a Ca2+

reservoir readily accessible for Ca2+-inducedCa2+ release in cardiac muscle, andpresumably for depolarization-induced Ca2+

release in skeletal muscle as well. Ca2+

release from the SR during excitation-contraction (E-C) coupling is a very fastprocess, especially in skeletal muscle, whichis completed in the ms time range. Mostluminal SR Ca2+ is bound, and thus prior torelease it must first dissociate from CSQ.Hence, it becomes relevant to determine thekinetics of Ca2+ dissociation from CSQ, andalso to determine how fast CSQ undergoesconformational changes following Ca2+

dissociation, since these changes may affectRyR activity. In fact, in response to RyRactivation by caffeine or polylysine, CSQreleases Ca2+ to the SR lumen before Ca2+ isreleased from the SR (Ikemoto et al., 1991).These results suggest that RyR activation issensed by CSQ and raise the intriguingpossibility that CSQ may transmit to RyRconformational changes associated with Ca2+

dissociation, modifying their activity. Yet,there are no direct measurements on the rateof Ca2+ dissociation from CSQ, so it is notknown whether this process is faster than E-C coupling or whether it becomes ratelimiting. Accordingly, the aim of the presentwork was to measure the kinetics of Ca2+

dissociation from CSQ still attached to thejunctional SR membrane or in solution.

BELTRÁN ET AL. Biol Res 39, 2006, 493-503496

METHODS AND MATERIALS

Isolation procedures

Triad-enriched SR vesicles were isolatedfrom rabbit white skeletal muscle as reportedpreviously (Hidalgo et al., 1993). Usingthese vesicles as starting material, CSQ waspurified as described (Cala and Jones, 1983).Junctional face membranes (JFM) wereisolated as reported elsewhere (Costello etal., 1986), with some minor modifications.Briefly, Triton X-100 was added at a finalconcentration of 0.5% to vesicles pre-incubated at 4ºC for 10 min in a solutioncontaining 0.3 M sucrose, 1 mM CaCl2, 20mM MOPS-Tris, pH 6.8. This mixture wasincubated on ice for 20 min, with vigorousstirring for 15 s in a Vortex mixer every 5min and was sedimented at 48,000 x g for 1h. The resulting pellet was resuspended in0.3 M sucrose, 1 mM CaCl2, 20 mM MOPS-Tris, pH 6.8, frozen at -80ºC and used withina week after freezing. To prepare JFMwithout CSQ, solid Tris base and EGTA(from a stock solution of 100 mM) wereadded to thawed JFM fractions to reach pH8.0 and a final concentration of 1 mMEGTA. The mixture was incubated at 4ºC for10 min, sedimented at 48,000 x g for 1 h,and the resulting pellet was resuspended in0.3 M sucrose, 1 mM CaCl2, 20 mM MOPS-Tris, pH 6.8, frozen and used as above.

Determination of the kinetics of calciumdissociation from CSQ or JFM

All kinetic experiments were done at 25ºC ina SX.18MV fluorescence stopped-flowspectrometer from Applied PhotophysicsLtd. (Leatherhead, UK). Calciumconcentration changes were monitored aschanges in Calcium Green-2 fluorescence,with λex = 506 nm and using a 530 nmcutoff filter (Oriel, Stratford, CT). Tomeasure calcium dissociation kinetics fromCSQ in solution, purified CSQ wasequilibrated at 0.11 mg/ml in a solutioncontaining 1.0 mM CaCl2, 0.1 M KCl, 20mM Tris/MOPS, pH 7.2. At time zero, 10volumes of this solution were mixed in thestopped flow spectrometer with one volumeof a solution containing 13.2 mM BAPTA, 1

mM Calcium Green-2, 0.1 M KCl, 20 mMMOPS/Tris, pH 7.2. The resulting free[Ca2+] was calculated as 0.64 μM using theWinMaxC program (www.stanford.edu/-cpatton/winmaxc2.html). To measurecalcium dissociation from JFM, membranes(3.3 mg/ml) were dialyzed for 36 h at 4ºC ina solution containing 2.2 mM CaCl2, 0.1 MKCl, 20 mM MOPS-Tris, pH 7.2. At timezero, 1 volume of this solution (previouslywarmed to room temperature) was mixed inthe stopped flow spectrometer with 10volumes of a solution containing 0.286 mMBAPTA, 1 μM Calcium Green-2, 0.1 MMOPS-Tris, pH 7.2. The resulting free[Ca2+] was calculated as above as 0.67 μM.

Determination of the kinetics of CSQ orJFM intrinsic fluorescence changes

Intrinsic fluorescence changes weremonitored with λex =295 nm and a 320 nmcutoff filter (Oriel, Stratford, CT). Tomeasure the kinetics of intrinsic fluorescencechanges that take place following the mixingof calcium-equilibrated CSQ with a calcium-free solution, purified CSQ was equilibratedat a final concentration of 0.11 mg/ml in asolution containing 1.0 mM CaCl2, 0.1 MKCl, 20 mM Tris/MOPS, pH 7.2. At timezero, 10 volumes of this solution were mixedin the stopped-flow spectrophotometer withone volume of a solution containing 13.2mM BAPTA, 0.1 M KCl, 20 mM MOPS/Tris, pH 7.2; after mixing, the calculatedinitial free [Ca2+] was 0.64 μM. To measurethe kinetics of intrinsic fluorescence changesthat take place after mixing calcium-equilibrated JFM with a calcium-freesolution, membranes (3.3 mg/ml) weredialyzed as above in a solution containing2.2 mM CaCl2, 0.1 M KCl, 20 mM Tris/MOPS, pH7.2. At time zero, 1 volume ofthis solution was mixed in the stopped flowspectrometer with ten volumes of a solutioncontaining 0.286 mM BAPTA, 0.1 M KCl,20 mM MOPS/Tris, pH 7.2, which gives acalculated free [Ca2+] of 0.67 μM.

Other procedures

SDS-PAGE was carried out according toLaemmli (1970). Gels were stained with

497BELTRÁN ET AL. Biol Res 39, 2006, 493-503

Coomassie blue or Stains-All (Campbell etal. , 1983). Protein concentration wasdetermined (Hartree, 1972) usingcommercial bovine serum albumin asstandard.

Materials

All reagents used were of analytical grade.Bovine serum albumin, Coomassie Blue,Stains-All, and protease inhibitors(Leupeptin, Pepstatin A, benzamidine, andPhenylmethylsulfonyl fluoride) were fromSigma Chemical Co. (St. Louis, MO, USA).Calcium Green-2 was obtained fromMolecular Probes (Eugene, OR, USA) andTriton X-100 from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA).

RESULTS AND DISCUSSION

Calcium dissociation from purified CSQ insolution

We used Calcium Green-2 to measurechanges in solution Ca2+ following Ca2+

dissociation from purified CSQ. Asillustrated in Figure 1, in the presence ofSR vesicles, Calcium Green-2 displayed aKd for Ca2+ binding of 0.98 μM.

A solution containing purified CSQequilibrated in 1 mM [Ca2+] was mixed 10:1 at time zero with a solution containingCalcium Green-2 and 13.2 mM BAPTA.After mixing, initial [Ca2+] (calculated as0.64 μM) increased rapidly following amono-exponential function with k = 11.6 s-1

(Fig. 2A, trace a). Dilution of the above twosolutions without CSQ did not produce asignificant fluorescence change (Fig. 2A,trace b). Considering that the CSQconcentration after mixing was 2 μM andassuming that at 1 mM [Ca2+] plus 0.1 MKCl CSQ is 50% saturated with calcium (25mol Ca2+/mol CSQ), it can be estimatedthat solution [Ca2+] should have increasedto about 50 μM. But the presence ofBAPTA (final concentration 1.2 μM) isexpected to decrease [Ca2+] to 0.81 μM,enough to induce complete dissociation ofcalcium from CSQ without inducingsaturation of Calcium Green-2 fluorescence(see Fig. 1).

Figure 1. Determination of the Kd of Ca2+ binding to Calcium Green-2. Calcium Green-2fluorescence was measured with λex = 536 nm, λem = 506 nm. Solutions contained 90 nM CalciumGreen-2 in 0.1 M KCl, 20 mM MOPS-Tris, pH 7.2, 0.2 mg per ml of triad-enriched SR vesicles andvarying free [Ca2+], checked with a calcium electrode.

BELTRÁN ET AL. Biol Res 39, 2006, 493-503498

Intrinsic fluorescence changes of purifiedCSQ in solution

Parallel experiments revealed that CSQintrinsic fluorescence decreased rapidlyafter mixing, as above, CSQ equilibratedwith Ca2+ with the BAPTA containingsolution. The intrinsic fluorescencedecrease followed a mono-exponential

Figure 2. Kinetics of calcium dissociation (A) and intrinsic fluorescence changes (B) for purifiedcalsequestrin in solution. A: Values of Calcium Green-2 fluorescence averaged from 8 independenttraces are shown. In a, data obtained after mixing a solution containing calsequestrin equilibrated withCa2+ with a Ca2+-free solution followed a single exponential function with k = 11.6 s-1. In b, datacorrespond to mixing the same solutions as above, except that the Ca2+-containing solution was devoidof calsequestrin. B: Conditions were the same as in A, except that fluorescence was determined with λex

= 295 nm to follow intrinsic fluorescence changes. Values, averaged from 5 independent traces,followed a single exponential function with k = 6.7 s-1. For further details, see text.

function, with k = 6.7 s-1 (Fig. 2B, trace a).Mixing just the two solutions, withoutCSQ, did not produce a significant changein fluorescence (Fig. 2B, trace b).

These combined results suggest thatCa2+ dissociation from purified CSQ insolution is a faster process (t1/2 = 60 ms)than the changes in intrinsic fluorescence(t1/2 = 103 ms).

499BELTRÁN ET AL. Biol Res 39, 2006, 493-503

Calcium dissociation from CSQ formingpart of junctional face membranes

Inside the SR lumen, CSQ forms a networkand is anchored to the JFM (Franzini-Armstrong et al., 1987; Maguire et al.,1997), presumably through its interactionswith triadin and junctin. Due to the SRpermeability barrier to calcium, directdetermination of Ca2+ dissociation fromCSQ present in the lumen of intactjunctional SR vesicles with anextravesicular Ca2+ indicator is not possiblein our conditions. Thus, to compare thedissociation rate of Ca2+ from CSQ inconditions close to the physiologicalsituation, in which CSQ is present at highdensity, we measured Ca2+ dissociationfrom CSQ forming part of JFM. The JFMfraction has CSQ still attached to it butdoes not represent a permeability barrier.Accordingly, direct measurements of Ca2+

dissociation kinetics are possible with ourstopped flow system.

As illustrated in a gel stained withCoomassie blue (Fig. 3, top panel), the JFMfraction (lane 5) is significantly enriched inCSQ when compared to the SR vesicles(lane 1); the JFM fraction is practicallydevoid of Ca-ATPase, which is present inthe Triton X-100 supernatant (lane 3).Incubation of JFM with EGTA resulted inextraction of CSQ from the JFM (lane 4) tothe EGTA-supernatant (lane 2). The lowerpanel of Figure 3 shows the same gel afterremoving the Coomassie blue stain andstaining with Stains-All, which stains bluecalcium-binding proteins such as CSQ andpink all other proteins.

A solution containing 3.3 mg per ml ofJFM previously equilibrated with 2.2 mMCaCl2 for 36 h (see Materials and Methods)was mixed 1: 10 with a solution containingCalcium Green-2 plus 0.286 mM BAPTA.Following mixing, protein concentrationwas 0.3 mg/ml, total Ca2+ was 0.2 mM, andinitial free [Ca2+] was 0.67 μM; solution[Ca2+] increased rapidly following a mono-exponential function with k = 106.1 s-1

(Fig. 4A, trace a). Dilution 1: 10 of asolution containing 3.3 mg per ml of CSQ-free JFM previously equilibrated with 2.2mM [Ca2+] with a solution containing 0.286

mM BAPTA did not produce a significantincrease in Calcium Green-2 fluorescencebut caused a very small and fastfluorescence decrease that was completedin less than 10 ms (Fig. 4A, trace b).

Upon mixing, the final JFMconcentration became 0.3 mg/ml, of whichabout 50% corresponds to CSQ (Fig. 3)giving a final CSQ concentration of 3 μM.Assuming that at 2 mM [Ca2+] CSQ was67% saturated with Ca2+ (33 mol/mol), itcan be estimated that solution [Ca2+] shouldhave increased by about 100 μM. Butconsidering BAPTA (final concentration0.26 mM), it can be calculated that solution[Ca2+] increased only to 4.1 μM. Again, thisCa2+ range is enough to induce completedissociation of calcium from CSQ withoutinducing saturation of Calcium Green-2fluorescence (Fig. 1).

In parallel experiments, mixing the samesolutions as above revealed that theintrinsic fluorescence of JFM decreasedrapidly after mixing JFM equilibrated withCa2+ with the BAPTA-containing solution(Fig. 4B, trace a). In this case, thefluorescence decay was not well adjustedby a single exponential function (brokenline, Fig. 4B, inset) but was better adjustedby a double-exponential function with k1 =54.8 s-1 and k2 = 7.4 s-1. The magnitude ofeach exponential component was aboutequal. It is noteworthy that the k2 valuewas not significantly different from the kvalue of the intrinsic fluorescence changeexhibited by CSQ in solution. Similardilution of CSQ-free JFM with the BAPTA-containing solution produced only a smallfluorescence decrease (Fig. 4B, trace b),suggesting that essentially all intrinsicfluorescence changes displayed by JFMwere contributed by CSQ.

A summary of values obtained in severalexperiments is given in Table 1. The halftime of calcium dissociation from CSQforming part of the JFM was ≤ 6.4 ms, at25ºC. It is likely that calcium dissociationwill be even faster at 37ºC. These resultsimply that calcium dissociation from CSQin vivo is not rate limiting for calciumrelease from the SR.

The results summarized in Table 1 alsoindicate that calcium dissociation from

BELTRÁN ET AL. Biol Res 39, 2006, 493-503500

CSQ, either in solution or forming part ofthe JFM, was faster than the associatedintrinsic fluorescence changes. Theseresults indicate that the structuralrearrangements of CSQ that follow Ca2+

dissociation and that originate the intrinsicflorescence decrease occur with some delayafter Ca2+ dissociation. Noteworthy, Ca2+

dissociation from CSQ and the subsequentintrinsic fluorescence changes were faster

Figure 3. Protein composition of JFM as determined by SDS-electrophoresis. Top panel, 2-12%polyacrylamide gradient gel stained with Coomassie Blue. Lower panel, the same gel, after removalof the Coomassie Blue stain and staining with Stains All. Lane 1: Triad-enriched SR vesicles. Lane2: Supernatant of JFM after CSQ extraction. Lane 3: supernatant of SR vesicles extracted withTriton X-100 to prepare JFM. Lane 4: JFM devoid of CSQ. Lane 5: JFM.

when CSQ was still attached to JFM than insolution. It has been reported that CSQ ispresent in the SR lumen in a wide range ofhigh molecular mass clusters (Maguire etal., 1997). Thus, our findings suggest thatwhen present at high concentrations in arestricted space, such as when attached tothe JFM, CSQ may also form clusters fromwhich calcium would dissociate in acooperative fashion.

501BELTRÁN ET AL. Biol Res 39, 2006, 493-503

Figure 4. Kinetics of calcium dissociation from JFM (A) and the associated intrinsic fluorescencechanges (B). A: Values of Calcium Green-2 fluorescence averaged from 4 independent traces areshown. In a, data obtained after mixing JFM equilibrated with Ca2+ with a Ca2+-free solutionfollowed a single exponential function with k = 106.1 s-1. In b, data correspond to mixing the samesolutions as above, except that the Ca2+-containing solution contained JFM devoid of CSQ. B:Conditions were the same as in A, except that fluorescence was determined with λex = 295 nm tofollow intrinsic fluorescence changes. In a, values from JFM and averaged from 4 independenttraces followed a double exponential function with k1 = 54.8 s-1 (55.4% of the total fluorescencechange) and k2 = 7.4 s-1 (44.6% of the total fluorescence change). Values in b correspond to CSQ-free JFM. For further details, see text.

BELTRÁN ET AL. Biol Res 39, 2006, 493-503502

In response to RyR activation by sub-maximal concentrations of releasing agents,CSQ unbinds Ca2+ (and presumablyundergoes a significant change inconformation, as determined here) beforeCa2+ is released from the SR (Ikemoto etal., 1991). These results raise the possibilitythat the E-C coupling signal transmittedfrom the transverse tubule voltage sensorsto RyR channels also may be sensed byCSQ, resulting in fast Ca2+ dissociationfrom CSQ. As discussed above, followingCa2+ dissociation, CSQ would loose its α-helical content to adopt a random coilconformation that would inhibit RyRactivity. If this mechanism operates duringE-C coupling, CSQ via Ca2+-dependentconformational changes may control howlong RyR channels remain open afteractivation. Clearly, more experimentalevidence is needed to investigate in furthermolecular detail how CSQ contributes toregulate Ca2+ release during E-C couplingin skeletal and cardiac muscle.

ACKNOWLEDGEMENTS

Paulina Donoso’s critical reading of thismanuscript is gratefully acknowledged.This work was supported by FONDAPCenter for Molecular Studies of the Cell,Fondo Nacional de Investigación Científicay Tecnológica (FONDECYT) 15010006.

REFERENCES

BEARD NA, SAKOWSKA MM, DULHUNTY AF,LAVER DR (2002) Calsequestrin is an inhibitor ofskeletal muscle ryanodine receptor calcium releasechannels. Biophys J 82: 310-320

BEARD NA, LAVER DR, DULHUNTY AF (2004)Calsequestrin and the calcium release channel ofskeletal and cardiac muscle. Prog Biophys Mol Biol85: 33-69

CALA SE, JONES LR (1983) Rapid purification ofcalsequestrin from cardiac and skeletal musclesarcoplasmic reticulum vesicles by Ca2+-dependentelution from phenyl-sepharose. J Biol Chem 258:11932-11936

CAMPBELL KP, MACLENNAN DH, JORGENSEN AO(1983) Staining of the Ca2+-binding proteins,calsequestrin, calmodulin, troponin C, and S-100, withthe cationic carbocyanine dye “Stains-all”. J BiolChem 258: 11267-11273

COSTELLO B, CHADWICK C, SAITO A, CHU A,MAURER A, FLEISCHER S (1986) Characterization ofthe junctional face membrane from terminal cisternae ofsarcoplasmic reticulum. J Cell Biol 103: 741-753

COZENS B, REITHMEIER RA (1984) Size and shape ofrabbit skeletal muscle calsequestrin. J Biol Chem 259:6248-6252

DONOSO P, PRIETO H, HIDALGO C (1995) Luminalcalcium regulates calcium release in triads isolatedfrom frog and rabbit skeletal muscle. Biophys J 68:507-515

DONOSO P, BELTRÁN M, HIDALGO C (1996) LuminalpH regulates calcium release kinetics in sarcoplasmicreticulum vesicles. Biochemistry 35: 13419-13425

FRANZINI-ARMSTRONG C, KENNEY LJ, VARRIANO-MARSTON E (1987) The structure of calsequestrin intriads of vertebrate skeletal muscle: A deep-etch study.J Cell Biol 105: 49-56

GUO W, CAMPBELL KP (1995) Association of triadinwith the ryanodine receptor and calsequestrin in thelumen of the sarcoplasmic reticulum. J Biol Chem 270:9027-9030

GYORKE S, GYORKE I, TERENTYEV D,VIATCHENKO-KARPINSKI S, WILLIAMS SC(2004) Modulation of sarcoplasmic reticulum calciumrelease by calsequestrin in cardiac myocytes. Biol Res37: 603-607

HARTREE EF (1972) Determination of protein: Amodification of the Lowry method that gives a linearphotometric response. Anal Biochem 48: 422-427

HE Z, DUNKER AK, WESSON CR, TRUMBLE WR(1993) Ca(2+)-induced folding and aggregation ofskeletal muscle sarcoplasmic reticulum calsequestrin.The involvement of the trifluoperazine-binding site. JBiol Chem 268: 24635-24641

HIDALGO C, JORQUERA J, TAPIA V, DONOSO P(1993) Triads and transverse tubules isolated fromskeletal muscle contain high levels of inositol 1,4,5-trisphosphate. J Biol Chem 268: 15111-15117

TABLE 1

Rate constants of calcium dissociation from calsequestrin and of the associated intrinsicfluorescence changes

Rate Constant of Rate Constant ofCalcium Dissociation, s-1 Intrinsic Fluorescence change, s-1

Calsequestrin in Solution 11.5 ± 0.3 (n = 9) 6.5 ± 0.1 (n = 5)

Junctional Face Membranes 109.0 ± 1.4 (n = 4) k1 = 54.12 ± 2.3 (55.4%)k2 = 6.88 ± 0.29 (44.6%) (n = 4)

Data represent Mean ± S.E. See text for further details.

503BELTRÁN ET AL. Biol Res 39, 2006, 493-503

HIDALGO C, DONOSO P, RODRÍGUEZ PH (1996)Protons induce calsequestrin conformational changes.Biophys J 71: 2130-2137

IKEMOTO N, BHATNAGAR GM, NAGY B, GERGELY J(1972) Interaction of divalent cations with the 55,000-dalton protein component of the sarcoplasmicreticulum. Studies of fluorescence and circulardichroism. J Biol Chem 247: 7835-7837

IKEMOTO N, RONJAT M, MESZAROS LG, KOSHITAM (1989) Postulated role of calsequestrin in theregulation of calcium release from sarcoplasmicreticulum. Biochemistry 28: 6764-6771

IKEMOTO N, ANTONIU B, KANG JJ, MESZAROS LG,RONJAT M (1991) Intravesicular calcium transientduring calcium release from sarcoplasmic reticulum.Biochemistry 30: 5230-5237

LAEMMLI UK (1970) Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature 227: 680-685

LAMB GD, CELLINI MA, STEPHENSON DG (2001)Different Ca2+ releasing action of caffeine anddepolarisation in skeletal muscle fibres of the rat. JPhysiol 531: 715-728

MACLENNAN DH, WONG PT (1971) Isolation of a

calcium-sequestering protein from sarcoplasmicreticulum. Proc Natl Acad Sci USA 68: 1231-1235

MAGUIRE PB, BRIGGS FN, LENNON NJ,OHLENDIECK K (1997) Oligomerization is anintrinsic property of calsequestrin in normal andtransformed skeletal muscle. Biochem Biophys ResCommun 240: 721-727

MURRAY BE, OHLENDIECK K (1998) Complexformation between calsequestrin and the ryanodinereceptor in fast- and slow-twitch rabbit skeletal muscle.FEBS Lett 429: 317-322

ROJAS E, HIDALGO C (1968) Effect of temperature andmetabolic inhibitors on 45Ca outflow in squid giantaxons. Biochim Biophys Acta 163: 550-556

WANG S, TRUMBLE WR, LIAO H, WESSON CR,DUNKER AK, KANG CH (1998) Crystal structure ofcalsequestrin from rabbit skeletal muscle sarcoplasmicreticulum. Nat Struct Biol 5: 476-483

ZHANG L, KELLEY J, SCHMEISSER G, KOBAYASHIYM, JONES LR (1997) Complex formation betweenjunctin, triadin, calsequestrin, and the ryanodinereceptor. Proteins of the cardiac junctionalsarcoplasmic reticulum membrane. J Biol Chem 272:23389-23397

BELTRÁN ET AL. Biol Res 39, 2006, 493-503504