Silver Ions Trigger Ca2+ Release by Interaction with the (Ca2+-Mg2+ ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 262. No. 16. Issue of June 5,pp. 76767679,1987 Printed in U.S.A.

0 1987 by The American Society of Biological Chemists, Inc.

Silver Ions Trigger Ca2+Release by Interaction with the (Ca2+-Mg2+)-ATPase in Reconstituted Systems* (Received for publication, November 25,1986)

Gwyn W. Gould, John Colyer, J. Malcolm East$, and Anthony G. Lee From the Department of Biochemistry, University of Southampton, Southumpton, SO9 3TU, United Kingdom

It has been suggested that vesicles derived from the other reagents known t o react withsulfhydrylgroups will sarcoplasmic reticulum of skeletal muscle contain Ca2+ produce a dramatic increase in Caz+ efflux from SR vesicles channels which can be opened by interaction with and it hasbeen suggested that this occurs through binding to sulfhydryl reagents such as Ag+ or Hg". We show that, sulfhydryl groups on Ca'+ channels within the SR membrane, in reconstituted vesicles containing the (Ca2+-Mg2+)- resulting in openingof the channels(6-8). ATPase purified from sarcoplasmic reticulum as the Importantly, it isnoticeable that agentswhich modify Ca'+ only protein, the ATPase can act as a pathway for Ca2+ release through the proposed Ca2+ channel also modify the efflux and that Ag+ induces a rapid release of Ca2+ activity of the (Ca2+-Mg2')-ATPase. Thus, pH, M$+, Ca2+, from such reconstituted vesicles. We also show that adenine nucleotides, carbodiimides, and amine-based drugs, Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the all of which affect Ca'+ efflux, also affect ATPase activity.' (Ca2+-Mg2+)-ATPase can act as a pathway for rapid We have, therefore, suggested that Ca2+efflux from SR vesicles could be mediated by the ATPase, using the same ca'+ CaZ+release from sarcoplasmic reticulum. binding sitesas are used in Ca2+uptake. We haveshown that Ca2+effluxcould be explainedquantitatively in terms of of the (Ca2+-Mg2+)Scheme 1.' Studies of the ATPase activity The sarcoplasmic reticulum (SR)' of skeletal muscle is ATPase have suggested that the ATPase can exist in oneof responsible primarily for the regulation of the cytosolic free two conformations, E l or E2. In the El conformation, the Ca'+ concentration. Removal of Ca2+ fromthe cytosol to cause two Ca2+binding sites/ATPase molecule are of high affinity muscle relaxation occurs via the (Ca2+-Mg2')-ATPase which and are exposed on the outer (cytoplasmic) side of the SR is the major protein component of the SR membrane. The membrane. After phosphorylat,ion of the ATPaseby MgATP, mechanism of action of this ATPase is well established and the ATPase undergoes a conformation change in which the recently a comprehensive kinetic model for the ATPase was Ca2+binding sites become of low affinity and are exposed to presented (1). The processes underlying Ca2+release are, the inside of the SR (1). To explain Ca" effluxfrom SR however, much less well understood. Contraction is initiated vesicles mediated by the ATPase we suggested that, after by a neural impulse resulting in depolarization of the sarco- binding of Ca2+ to El, the ATPase can undergo a slow conlemma (the surface membrane of the muscle cell) and of its formational change inwhich the Ca2+binding sites transform invaginations (the T-system) and, presumably, this depolari- to a low affinity state characteristic of E2 but remainexposed zation initiates release of Ca2+ from the SR by electrical or on the outsideof the SR (state E3'Caz in Scheme 1).Cycling chemical coupling. Ca2+release from SR has been studied in of the Ca'' binding sites in this low affinity state between both skinned fibers and in fragmented SR vesicles and has being outward facing (E3'Ca') and inward facing (E4'Ca') been found to occur in response to a variety of stimuli ( 2 ) . then provides a pathway for Ca2+ efflux?We have shown that Thus, efflux of Ca2+ hasbeen observedfrom SR vesicles it was possible to interpret the Caz+ dependence of passive passively loaded with Ca2+ after dilution intomedia contain- Ca2+efflux in terms of Scheme 1, assuming the same paraming low concentrations of Ca'+ (3-5), and it has been suggested eters forCa2+binding to the conformational states E3 and E4 that this release occurs through Ca'+ release channels within as were derived previously forCa2+ binding E2 to from studies the SR membrane (3-5). Although with suitable concentra- on the inhibition of ATPase activity by high concentrations tions of Ca", M$+, and adenine nucleotides, passive efflux of Ca".' from SR vesicles can berelatively fast, it is not clear that the Of course, if the ATPase can indeed act as a pathway for rate of release is sufficiently fast under the conditions likely passive Ca2+efflux, then Ca2+efflux should beobserved from to be foundin a muscle cell toexplainthe Ca2+release reconstituted membrane vesicles containing the ATPase as responsibleformuscle contraction (2-5)? However, it has the only protein species and the characteristicsof this efflux been reported that low concentrations of mercury, silver and * This work was supported by the Science and Engineering Research Council and theWellcome Trust, a Medical Research Council studentship (to G . W. G.), and a Science and Engineering Research Council studentship (to J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Wellcome Trust Lecturer. ' The abbreviations used are: SR, sarcoplasmic reticulum; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, * J. M. McWhirter, G . W. Gould, J. M. East, and A. G . Lee (1987) Biochem. J.,in press.

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SCHEME 1. Proposed schemefor the Ca2+release pathway.2 In the E l conformation, the two CaZ+binding sitesper ATPase molecule are of high affinity and outward facing, whereas in the E3 conformation they are of low affinity and outward facing, and in the E2 and E4 conformations they are of low affinity and inward facing. The parameters describing the binding of Ca2+to E2, E3, and E4 are equal.'

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Calcium Release in Reconstituted Systems should mirror those observed for Ca'+ efflux from SR vesicles. We have indeed observed that the ATPase alone can mediate a specific release of Ca2+ from reconstituted vesicles with a sensitivity to pH, Ca'+, M$+, and adenine nucleotides comparable to thatobserved for SR vesicles.' Here we show that the effects ofAg' ionson Ca'+ efflux fromreconstituted vesicles and from SR vesicles are also comparable.

a b

7677

c d

-205 -1 16 97

MATERIALS AND METHODS

(Ca2+-M$+)-ATPasewas prepared from female rabbit (New Zealand White)hind leg muscle as described in East andLee (9). Briefly, this consisted of mincing about 500 g of muscle, followed by homogenization in a Waringblender for 30 s with 1.5 volumes of cold 0.3 M sucrose, 20 mM histidine, 1 mM dithiothreitol, and 5 p M phenylmethanesulfonyl fluoride (pH 8.0). All procedures were carried out a t 4 "C. The homogenate was centrifuged (8,000 X g, 15min). The undisrupted material was retreated as above, and the pooled supernatants were filtered through muslin and centrifuged (37,000 X g, 80 min). The pellets were resuspended in 150 ml of cold 0.3 M sucrose, 10 mM histidine, 0.6 M KCl, 1 mM dithiothreitol, and 5 p~ phenylmethylsulfonyl fluoride (pH 8.0) and left on ice for 30 min followed by centrifugation (5,000 X g, 20 min). The supernatant was carefully decanted from the soft actomyosin pellet and centrifuged (37,000 X g, 90 min). The resulting pellet was resuspended in 3-5 ml of 0.25 M sucrose, 1 mM KCl, and 50 mM potassium phosphate (pH 8.0). This buffer was used in all subsequent procedures, unless otherwise stated. The homogenate was dialyzed overnight against 1liter of buffer. The resulting SR vesicles (protein concentration>30 mg/ml) were treated with potassium cholate to give a final ratio of 0.4 mg of cholate/mg of protein. The solubilized SR was then loaded onto a discontinuous sucrose gradient (20/30%) with a 60% cushion, in 1 M KC1 and 50 mM potassium phosphate (pH 8.0) and centrifuged overnight (95,000 X g). The pure ATPase was collected from the 30-60% interface, resuspended in buffer, and centrifuged (95,000 X g, 60 min). The pellet was resuspended in buffer and dialyzed overnight against 1 liter of buffer containing 10 g of washed Amberlite XAD-2 ion exchange resin, and was aliquoted, frozen in liquid nitrogen, and stored a t -20 "C. Sodium dodecyl sulfate-polyacrylamide gels were run using the method of Laemmli and Faure (IO) with 10% running gels and 4% stacking gels and stained with Coomassie Blue R-250. Gels were also stained with Stains-all following the procedure of Campbell et al. (11).Gels were scanned using a Joyce-Loebl gel scanning apparatus. Gels were also blotted onto nitrocellulose paper and stained with colloidal gold following the procedure of Moeremans et al. (12). Egg yolk phosphatidylcholine and egg yolk phosphatidylethanolamine were obtained from Lipid Products. Sealed, reconstituted vesicles were obtained as follows. Lipid (10 mg) was dispersed into 600 pl of buffer (40 mM Hepes-KOH, pH 7.2) by vortex mixing for 15-30 s. An aliquot of 10% (w/v) potassium cholate was then added to give the required cho1ate:lipid ratio, usually 1 mg of cholate, 1 mg of lipid. The suspension was sonicated to clarity under nitrogen in a bath sonicator. An aliquot (3-10 pl) of 10% (w/v) deoxycholate was added to an aliquot of the purified ATPase (0.3-1.0 mg of protein in 15-50 pl of buffer) to give a final deoxycho1ate:ATPase ratio of 0.6:l.O mg. The mixture was vortexed for 5 s and then spun at 10,000 X g for 5 min to remove any unsolubilized aggregates. The lipid and protein samples were then mixed to give the required molar ratio of lipid to ATPase (usually 3,OOO:l). The detergent was then removed with Sephadex G-50 using the centrifugation method of Penefsky (13). Ca2+uptake by the reconstituted vesicles was followed a t 30 "C by dual wavelength spectrophotometry using arsenazo 111 to monitor the external Ca2+ concentration. Reconstituted vesicleswere added to buffer (40 mM Hepes-KOH, 100 mM KCl, 5 mM MgS04, pH 7.2) containing 50 p~ calcium chloride and 50 p M arsenazo 111, to give a final protein concentrationof 20-40 pglml. Ca2+ uptakewas initiated by addition of an aliquot of ATP in buffer at pH 7.2, to give a final ATP concentration of 0.5 mM. Uptake of Ca2+ was followed by measuring the change in absorbance a t 675-685 nm, using a Shimadzu UV3000 dual wavelength spectrophotometer. Ca2+efflux from reconstituted vesicles was determined using 4sCa2+. Vesicles were loaded with 45Ca2+by including 45Ca2+in the buffer used for reconstitution. Efflux was initiated by diluting the loaded vesicles 15-fold into buffer not containing Ca2+.Aliquots were then taken at theappropriate times and layered onto columns of Bio-Rad 50W-X8 resin (Tris form), previously equilibrated with 40 mM Hepes-

66

45

29

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified ATPase ( b and c ) and SR vesicles (aand d ) stained with Coomassie Blue (c and d ) or withcolloidal gold after transfer to nitrocellulose paper (aand b). KOH, 0.25 M sucrose, 10 mg/ml bovine serum albumin (pH 7.2). Vesicles werewashed through the column with bovine serum albumincontaining buffer and counted. ATPase activity was measured using the coupled enzyme assay described by East and Lee (9). RESULTSANDDISCUSSION

The most definitive procedure by which a particular membranefunctioncan beassigned to a particular membrane is in a protein is to demonstrate that that function expressed reconstituted membrane system containing the protein as the only protein species. T o study the role of the (Ca2+-Mp2+)ATPase of sarcoplasmic reticulum, we have purified the ATPase and reconstituted it intoa variety of membrane systems. Polyacrylamide gels of the purified ATPasestained with Coomassie Blue suggest that the ATPase is essentially pure

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Calcium Release in Reconstituted Systems loo

15

r

10

0

2

6

4

5

8 1 0 1 2

TIME (mins.)

FIG. 2. ATP-dependent Caz+ uptake followed by Ag+-induced CaZ+ release from reconstituted vesicles. Ca2+ uptake (nmol/mg of protein) and release were monitored by measuring extravesicular Ca2+through changes in the absorption of arsenazo 111. The ATPase was reconstituted in a mixture of egg yolk phosphatidylethanolamine and egg yolk phosphatidylcholine (molar ratio of 4:l) at a lipidprotein molar ratio of 3000:l. Vesicles were reconstituted in 40 mM Hepes-KOH, 100 mMKC1 (pH 7.2) and uptake was assayed in 40 mM Hepes-KOH, 100 mM KCI, 5 mM MgSOI, 50 PM Ca2+,at 30 "C. Uptake was initiated by addition of 0.5 mM ATP, and AgN03 was added 8 min later.

t

> 3

c U

a

0

2

4

6

8

1

0

CpMI FIG. 4. Effect of Ag+ on ATPase activity. ATPase activities for the purified ATPase ( A )and SR vesicles ( B )were measured in 40 AgNO,

0

0

2

4

TIME

6

8

1

0

(mlns. 1

FIG. 3. The effect ofAg+ on Ca2+ efflux from passively loaded reconstituted vesicles. The ATPase was reconstituted with

mM Hepes-KOH, 100 mM KCI, 5 mM M F , 2.0 mM ATP (pH 7.2), 37 "C, at theoptimal free Ca2+concentration, as a function of AgN0, concentration (PM).

lipid mixture used in the reconstitution and increases with increasing phosphatidylethanolamine content in mixtures of phosphatidylethanolamine and phosphatidylcholine. Although levels of Ca2+ accumulation are considerably higher when the vesicles contain a Ca'+-precipitating agent such as phosphate or oxalate, accumulation doesalsooccur in the absence of any precipitating agent.' Fig. 2 illustrates Ca'+ accumulation by vesicles reconstituted with a 4:l molar ratio (>97%) (Fig. 1).Gels transferred to nitrocellulose paper and of phosphatidylethanolamineto phosphatidylcholine, at a stained with colloidal gold show the presence of a number of total 1ipid:protein molar ratio of 3000:1, in the absence of any lower molecular weight bands, together with the ATPase (Fig. Ca2+-precipitating agent. As shown, addition of 3 or 10 PM 1).A number of these bands can be assigned to breakdown AgN03 to thevesicles following Ca'+ accumulation leads toa products of the ATPase since they bind tomonoclonal anti- rapid release of the accumulatedCa'+. The concentrations of bodies raised to the purified ATPase.3 It is well known that Ag+ causing release ofCa'+ from the reconstituted vesicles the efficiency of transfer of low molecular weight proteins are the same as those causing release from native SR vesicles from gels to nitrocellulose paper is greater than that for high (7, 8). molecular weight proteins (15) so that the gold-stained gels Ca2+efflux fromreconstituted vesicles passively loaded with in Fig. 1 also indicate the lack of any major protein contami- 45Ca2+can be demonstrated by dilution of the vesicles into nant in the purified ATPase;thisisparticularlyclearin media free of Ca2+. In this way the rate ofCa'+ efflux has comparisons of gels of the purified ATPase and of native SR been shown to be sensitive to the lipid composition of the vesicles (Fig. 1).Gels stained with Stains-allalso fail to show vesicles, with the rateof Ca2+ efflux decreasing with increasing any major contaminant in the purified ATPase (data not content of phosphatidylethanolamine.' Fig. 3 shows that Ca2+ shown). Abramson et al. (7) reported that addition of micromolar efflux from vesicles reconstituted with phosphatidylcholine concentrations of AgNO, to SR vesicles that had accumulated and passively loaded with6 mM 45Ca2+is relatively slow when Ca" in the presence of ATP led to a rapid release of all or diluted 15-fold to give an external Ca2+ concentrationof 0.4 shown that high external Ca2+concentrations part of the accumulatedCa'+. We observe a similar effect for mM: it has been inhibit Ca'+ efflux both from reconstituted systems' and from reconstituted vesicles containing the(Ca2+-Mg2+)-ATPaseas SR vesicles (4,5). However, as shown in Fig. 3, addition native the only protein (Fig. 2). As reported by Navarro et al. (14), the level of Ca'+ accumulated by reconstituted vesicles follow- of 5 W M AgN03 leads t o a very rapid efflux of Ca'+ from the ing addition of ATP is sensitive to the composition of the reconstituted vesicles. Abramson et al. (7) reported that AgN03 stimulates the ATPase activity of SR vesicles. As shown in Fig. 4, we also J. Colyer, A. G. Lee, and J. M. East, unpublished observations.

egg yolk phosphatidylcholine at a lipidprotein molar ratio of 30001 in 40 mM Hepes-KOH, 100 mM KCl, 6.0 mM "Ca2+ (pH 7.2) and then diluted 15-fold into 40 mM Hepes-KOH, 100 mM KC1 (pH 7.2), 22 "C. The Ca2+content of the vesicles (expressed as percent of that at time 0 ) , was determined as described above. 5 min following dilution, 5 PM AgN03 was added.

Calcium Release in Reconstituted Systems find a stimulation of ATPase activity of SR vesicles at low concentrations of Ag+(less than 3 p ~but) inhibition a t higher concentrations of Ag+. For the purified ATPase, however, we find only inhibition of ATPase activity on addition ofAg+ (Fig. 4).The biphasic effect of Ag+on the ATPase activity of SR vesicles can, therefore, be attributed to an increase in the permeability of the vesicles to Ca" at low Ag+concentrations, leading to an increase in ATPase activity for the vesicles, together with inhibition of ATPase activity due to a direct effect of Ag+ on the ATPase. Abramson et al. (7) and Salama and Abramson (8) have argued that the effects ofAg+ that they see on native SR vesicles are not due to interaction of Ag+ with the ATPase but rather aredue to interactions with a "Ca'+ release protein or Ca2+channel." The close parallel that we observe between effects of Ag+ ions on Ca2+efflux from native SR vesicles and from reconstituted vesicles containing the (Ca2+-Mg2')-ATPase as theonly protein, and themarked effects of Ag+ on the activity of the purified ATPase show, however, that Ag+ ions do interact with the ATPaseand that this interaction is sufficient to account for all the effects observed with native SR vesicles. We conclude, therefore, that experiments with Ag+ ions provide no evidence for the presence of distinct Ca" channels in the membranes of the SR vesicles, but rather are consistent with the idea that the(Ca2+-Mg2+)-ATPase can act as a pathway for Ca2+efflux, as suggested.' Although it is obvious that theinteractions with Ag+studied here can have no direct physiological significance, the fact that a sulfhydryl reagent can cause a very rapid efflux of Ca2+

7679

mediated by the ATPase, does suggestthat theATPase could act as apathway for rapid release of Ca2+from SR in muscle. If the ATPase were to act as a pathway for Ca2+release in this way, then the pathway wouldhave to be controlled, presumably by interaction with other protein components present in the SR membrane. REFERENCES 1.Gould, G. W., East, J. M., Froud,R. J., McWhirter, J. M., Stefanova, H. I., and Lee, A. G. (1986) Biochem. J. 2 3 7 , 217227 2. Endo, M. (1977) Physiol. Rev. 57, 71-108 3. Martonosi, A. N. (1984) Physiol. Reu. 6 4 , 1240-1320 4. Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374 5. Meissner, G., Darling, E., and Eveleth, J. (1986) Biochemistry 25, 236-244 6. Bindoli, A., and Fleischer, S. (1983) Arch. Biochem. Biophys. 22 1,458-466 7. Abramson, J. J., Trimm, J. L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1526-1530 8. Salama, G., and Abramson, J. (1984) J. Bid. Chem. 259, 1336313369 9. East, J. M., and Lee, A. G. (1982) Biochemistry 21, 4144-4151 10. Laemmli, U. K., and Faure, M. (1973) J. Mol. Bwl. 80,575-599 11. Campbell, K. P., MacLennan, D. H., and Jorgensen, A. 0.(1983) J. Biol. Chem. 258,11267-11273 12. Moeremans, M., Daneels, G., and deMey, J. (1985) Anal. Bwchem. 145,315-321 13. Penefsky, H. E. (1979) Methods Enzymol. 56,527-530 14. Navarro, J., Toivio-Kinnucan, M., and Racker, E. (1984) BWchemistry 23,130-135 15. Gershoni, J. M.,Davis, F. E., and Palade, G. E. (1985) Anal. Biochem. 144,32-40