Vol. 185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 15, 1992
Pages 713-718
ENHANCEMENT
OF Ca2+-INDUCED Ca2+ RELEASE IN CALPAIN RABBIT SKINNED MUSCLE FIBERS
TREATED
MasamitsuIino, Hiromi Takano-Ohmuro,Yoko Kawana andMakoto Endo Department of Pharmacology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan 1I3 Received
April
26,
1992
Summary
Calpain treatmentof rabbit skinnedmusclefibers resultedin proteolysisof junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analysesindicate that calpain cleaves off -130 kDa peptide from the N-terminus. After suchtreatment9Ca2+ capacity of the sarcoplasmicreticulum remainednormal and both C!a2+ and adenine nucleotide dependenceof Ca2+-induced Ca2+ releasemechanismwere retained. However, the Ca2+-activatedCa2+ releaserate was increasedby two fold after the proteolysis. The results suggestthe presenceof functional domainsin the junctional foot protein, and the Nterminusdomain controls the activity of the Ca2+channel without changing Ca2+ and nucleotide sensitivities. 0 1992Academic Press,Inc.
Junctional foot protein (JFP) or ryanodine receptor of skeletalmusclecells is now thought to carry the function of Ca2+ releasechannel in the sarcoplasmicreticulum (SR) (1,2,3). In excitation-contraction coupling, the Ca2+ channelsareregulated,in a still unknown manner,by the “voltage sensors”on the transversetubule membranewhich detect the arrival of action potentials. JFP also functions as Ca2+-inducedCa2+release(CICR) mechanismwhich is different in various properties from the voltage sensor-gatedmode (4). The primary sequenceof JPP has been deducedfrom its cDNA, and the 4 to 12 membranespanningsegmentshave been assumedto be present near the C-terminus to form Ca2+ conducting pore (5,6). Although the Ca2+ release channel is modulated by various agents(4), modulator siteson the JFP molecule are not known, except for conflicting predictions basedon the primary amino acid sequence(5,6). It hasbeen shown that calpain cleavesJFP to fragmentsof various sizesleaving other SR proteins intact (7). It was suggestedthat the cleaved molecules still retain channel activities.
ABBREVIATIONS:
JFP, Junctional foot protein; SR, Sarcoplasmicreticulum; CICR, C$+induced Ca2+ release; MS, methanesulfonate; EGTA, ethylenglycol-bis(b-aminoethyl ether) N,N,N’,N’-tetraacetic acid; PIPES, piperazine-N-N’-bis(2-ethanesulfonicacid); AMPOPCP, plymethylenadenosine5’-triphosphate; SDS, sodiumdodecyl sulfate.
713
0006-291X/92 $4.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in an-y form reserved.
Vol.
185,
However,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
because the previous study measured single channel currents after incorporation
of
single molecules in lipid bilayer, neither the size of incorporated molecule nor the location of the proteolytic fragment along the primary sequence was known.
We have studied CICR mechanism
in skinned fibers after calpain treatment, so that we can examine the properties of calpain treated JFP proteins in the mass under various conditions. Molecular size of the calpain digested JFP was estimated by electrophoresis and immunoblot analysis. METHODS Skinned fiber bundles were prepared from Skinned fibers and calpain treatment rabbit psoas muscle bundles (~2 mm wide) which had been treated with saponin (50 l@ml) for 60 min in a relaxing solution: (in mM) ATP, 4.76; MgMq, 5.54; KMs, 108.6; NaN3,20; PIPES, 20; pH 7.0. They were immersed in a rigor solution (KMs, 100, EGTA, 10; PIPES, 20; pH 7.0) before treatment with m-calpain (Sigma) in a digesting solution (KMs, 100; &MS;?, 2; PIPES, 20; pH 7.0). Fiber bundles were treated for 60 min or longer with 100 p.g calpain/ml usually at room temperature (22-24crJ). Skinned fiber bundles for the electrophoretic analysis contained 5-10 fibers and were -20 mm long. For the CICR measurements specimens contained a couple of fibers and were 2 mm in length. Measurement of CZCR rates A microfluorometric method (8) was used to measure CICR rates of the SR in skinned fibers mounted in a microcuvette. Since calpain cleaved regulatory proteins of the contractile system (9), calpain-treated skinned fibers went into irreversible contracture in the presence of MgATP even without C$+. Therefore, Ca2+ was passively loaded into the SR by reverse flux of Ca 2+ through CICR channels (10) for 60 s in the presence of 1 mM Ca2+ and 10 mM AMP. In calpain untreated fibers, the passively loaded caz” amounted about 35% of active loading in the presence of 4 mM MgATP at pCa 6.7 for 120 s. Amount of passive loading was the same when the loading duration was increased to 120 s, nor was dependent on the calpain treatment. After the Ca2+ loading both Ca2’ and AMP were washed away. Then as a test procedure, Ca2+ (buffered with 10 mM EGTA) was applied to induce CICR. C$+ and EGTA were then washed away and 40 pM fura- was introduced. 50 mM caffeine was then applied in the presence of fura- to thoroughly release remaining Ca2’ in the SR, of which amount was measured by the fluorescence intensity change at 500 nm with alternating excitation at 345 and 365 nm. The amount of Ca2+ released during the test procedure was estimated by comparing assays with and without the test procedure. Amount of Ca2+ in the SR declined almost exponentially to zero with increasing duration of the test procedure. Thus, the activity of CICR was expressed by the decay rate constant. Compositions of solutions and the further detail of the protocol are described elsewhere (8). Experiments were carried out at pH 7.0 and at room temperature. Electrophoresis and immunoblot analyses Proteins in skinned fibers were solubilized with 15 ~1 of solution containing 2% CHAPS, 2 mM EGTA, 0.1 mM (p-amidin phenyl) methane fluoride-HCl, 2 mM MgCl,, 200 mM NaCl, 20 mM potassium phosphate buffer (PH 7.0) for 60 min on ice. The extract was mixed with equal volume of a solution containing 5% SDS, 5% 2mercaptoethano1, and 0.1 M Tris-HCl buffer (pH 6.8), and 15 ~1 of the mixture was loaded onto each gel lane. Proteins were transferred from SDS polyacrylamide gels to nitrocellulose membrane (1 1), which was then treated with 5% non-fat milk before incubation for at least 60 min with a 714
Vol.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
polyclonal antibody against the synthetic decapeptide corresponding to the C-terminus end sequence of rabbit JFP (5, 6). The antibody (EC-3) was raised in guinea pig. The immunocomplexwasvisualized usingthe horseradishperoxidasereaction.
RESULTS Fig. 1 showsthe immunoblot analysisof calpain treated shinnedfibers. The mobility of JFP reacting with the anti-C-terminus antibody increasedafter 60 to 90 min treatment with mcalpain, and becamealmost the sameasthat of dystrophin (not shown). We have not so far found smallerproteolyic fragmentsreacting with the antibody even when we carried out more extensive treatment:increaseof the treatmenttime up to 360 min, three-fold increaseof calpainconcentration, treatmentat 3793,or useof thinner bundleswith only a coupleof fibers to facilitate diffusion of the enzyme. Fig. 2 showsthe ratesof CICR measuredat pCa 6.5,6.0 and 5.5 in skinned fibers with or without calpain treatment. The difference in the Ca2+ releaserate was small at the lowest Ca2+ concentration, whereas the CICR rates were increased by about 2-fold at the higher Ca2+ concentrations. We alsocarried out experimentsunderquasi-physiologicalcondition, i.e. in the presenceof Mg2+ and adenine nucleotide. To avoid both reuptake of C$+ into the SR and contraction of fibers we used2 mM AMPOPCP, a nonhydrolyzable ATP analog, in place of ATP. As shown in
RR.
6.5
6.0 PCs
Fig. 1. Immunoblot analysis of calpain digested JFP. Polyclonal antibody against the C-terminus sequence was used to stain JFP of calpain untreated (right lane) and treated skinned fibers. Calpain treatment was carried out for 60 min (left) or 90 min (center). R.R.: ryanodine nxeptor or JFP. Fig. 2. Effect of calpain treatment on the rates of CICR in the absence of both Mg2+ and nucleotide. Means and SEM. * 0.01 < P < 0.02, ** P c 0.01 (r-test). 715
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.5 mM Mg, 2 mM AMPOPCP
F
.E a
Cl control E4 calpain
0.15
-
0.00
pCa 5.5
pCa 6.0
Fig. 3. Ratesof CICR in thepresence of 1.5mM Mg2+ and2 mM AMPOPCP with or without calpaintreatment. Numbersin the parentheses indicate the number of experiments. Means and SEM. ** P < 0.01.
Fig. 3 there was little effect of calpain treatmenton the rate of CICR at pCa 6, but 2-fold increase in the rate of Ca2+releasewas observedat pCa 5.5.
DISCUSSION The present study showsthat JFP is cleaved within muscle fibers by calpain to leave a polypeptide that includesthe C-terminus. Its mobility in SDS polyacrylamide gel was the sameas that of dystrophin whosemolecular massis -430 kDa (12). Since the molecularweight of JFP is -560 kDa (5,6), the cleavage site is located near residue 1200, and seemsto correspondto the protease-sensitiveregion I of JFP (13) (Fig. 4). This is probably one of the earliest proteolytic fragments of JFP in SR vesicle preparationsreported previously (7). In SR vesicles, JFP was further cleaved by calpain to about 150 kDa polypeptide. However, we have been so far unableto
Calpain clpvage site
I cl
* 1000 oorcine
N terminus domain Suppressor
I I II YY n
Surface oriented Im 2000
C terminus domain Ca channel Modulator sites
Fig. 4. Schematic diagram of the primary structure of JFP. Putative channel forming domains after two research groups (5,6) and surface oriented segments (12) are indicated, respectively, by the hatched bar and the boxes. The arrow indicates expected cleavage site of calpain. *Porcine malignant hyperthermia mutation is located at the 615th amino acid (14).
716
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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cleave the 430 kDa fragment further. This may suggest that other cleavage sites are protected in skinned fibers. Even after most of JFP were cleaved by calpain, the CICR mechanism of the skinned fibers retained its Ca2’ sensitivity,
although the maximum rate of release was doubled in qualitative
agreement with the results obtained in SR vesicles incorporated
in lipid bilayers (7).
In the
previous study it was impossible to determine the molecular size of the incorporated JFP, and the correlation between the peptide length and the function was not decisive. We have now shown that -430 kDa peptide seems to contain Ca 2+ channel and Ca2+ binding sites as well as the C-terminus. Furthermore, AMPOPCP.
we found essentially
the same effect of calpain treatment in the presence of
Therefore, it is likely that the adenine nucleotide binding site is also located in the C-
terminus peptide. On the other hand the N-terminus peptide may suppress the activity of the CICR channels. In porcine malignant hyperthermia, the CICR is enhanced in a very similar manner as in the calpain treated samples (14). Such effect has been attributed to a point mutation that causes change of 615th amino acid from arginine to cysteine (15). This site is located in the middle of the N-terminus peptide of the present study. This seems to be consistent with the notion that the Nterminus domain is involved in the suppression of channel activity. The present results indicate that JFP is composed of functional domains. The N-terminus domain seems to modulate the opening of Ca2+ channel without nucleotide sensitivities.
changing Ca2+ or adenine
It will be interesting to see if that domain is involved in the excitation-
contraction coupling of skeletal muscle.
ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan and from the National Center for Neurology and Psychiatry of the Ministry of Health and Welfare of Japan.
REFERENCES 1. 2. 3. 4. 5.
Hymel, L.M., Inui, S., Fleischer, S. and Schindler, H. (1988) Proc. Nat. Acad. Sci. USA 85, 441445. Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.-Y. and Meissner, G. (1988) Nature 331, 315-319. Imagawa, T., Smith, J.S., Coronado, R., Campbell, K.P. (1987) J. Biol. Chem. 262, 64606463. Endo, M. (1985) Curr. Top. Membr. Trunsp. 25, 181-230. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K. Minamino, N., Masuo, H., Ueda, M., Hanaoka, M., Hirose, T. and Numa, S. (1989) Narure 339, 439445. 717
Vol.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G. and MacLennan, D.H. (1990) J. Biol. Chem. 265,2244-2256. Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R. and Jones,L.R. (1990) Circ. Rex 67, 84-96. Iino, M. (1989) J. Gen. Physiol. 94, 363-383. Sugita, H., Ishiura, S., Suzuki, K. and Imahori, K. (1980) MuscleNerve 3, 335-339. Kitazawa, T. and Endo. M. (1976) Proc. JapanAcud 52,599~602. Reinnach, F.C., Masaki, T., Shafiq, T., Obinata, T. and Fishman, D.A. (1982) J. Cell. Biol. 95, 78-84. Koenig, M., Monaco, A.P. and Kunkel, L.M. (1988) Cell 53, 219-228. Marks, A.R., Fleischer, S. and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149. Ohta, T., Endo, M., Nakano, T., Morohoshi, Y., Wanikawa, K. and Ohga, A. (1989) Am . J. Physiol. 256, C358-C367. Fujii, J., Otsu, K., Zorzato, F., De Leon, S., Khanna, V.K., Weiler. J.E., O’Brien, P.J., MacLennan, D.H. (1991) Science253, 448-451.
718
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 15, 1992
Pages 713-718
ENHANCEMENT
OF Ca2+-INDUCED Ca2+ RELEASE IN CALPAIN RABBIT SKINNED MUSCLE FIBERS
TREATED
MasamitsuIino, Hiromi Takano-Ohmuro,Yoko Kawana andMakoto Endo Department of Pharmacology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan 1I3 Received
April
26,
1992
Summary
Calpain treatmentof rabbit skinnedmusclefibers resultedin proteolysisof junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analysesindicate that calpain cleaves off -130 kDa peptide from the N-terminus. After suchtreatment9Ca2+ capacity of the sarcoplasmicreticulum remainednormal and both C!a2+ and adenine nucleotide dependenceof Ca2+-induced Ca2+ releasemechanismwere retained. However, the Ca2+-activatedCa2+ releaserate was increasedby two fold after the proteolysis. The results suggestthe presenceof functional domainsin the junctional foot protein, and the Nterminusdomain controls the activity of the Ca2+channel without changing Ca2+ and nucleotide sensitivities. 0 1992Academic Press,Inc.
Junctional foot protein (JFP) or ryanodine receptor of skeletalmusclecells is now thought to carry the function of Ca2+ releasechannel in the sarcoplasmicreticulum (SR) (1,2,3). In excitation-contraction coupling, the Ca2+ channelsareregulated,in a still unknown manner,by the “voltage sensors”on the transversetubule membranewhich detect the arrival of action potentials. JFP also functions as Ca2+-inducedCa2+release(CICR) mechanismwhich is different in various properties from the voltage sensor-gatedmode (4). The primary sequenceof JPP has been deducedfrom its cDNA, and the 4 to 12 membranespanningsegmentshave been assumedto be present near the C-terminus to form Ca2+ conducting pore (5,6). Although the Ca2+ release channel is modulated by various agents(4), modulator siteson the JFP molecule are not known, except for conflicting predictions basedon the primary amino acid sequence(5,6). It hasbeen shown that calpain cleavesJFP to fragmentsof various sizesleaving other SR proteins intact (7). It was suggestedthat the cleaved molecules still retain channel activities.
ABBREVIATIONS:
JFP, Junctional foot protein; SR, Sarcoplasmicreticulum; CICR, C$+induced Ca2+ release; MS, methanesulfonate; EGTA, ethylenglycol-bis(b-aminoethyl ether) N,N,N’,N’-tetraacetic acid; PIPES, piperazine-N-N’-bis(2-ethanesulfonicacid); AMPOPCP, plymethylenadenosine5’-triphosphate; SDS, sodiumdodecyl sulfate.
713
0006-291X/92 $4.00 Copyright 0 I992 by Academic Press, Inc. All rights of reproduction in an-y form reserved.
Vol.
185,
However,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
because the previous study measured single channel currents after incorporation
of
single molecules in lipid bilayer, neither the size of incorporated molecule nor the location of the proteolytic fragment along the primary sequence was known.
We have studied CICR mechanism
in skinned fibers after calpain treatment, so that we can examine the properties of calpain treated JFP proteins in the mass under various conditions. Molecular size of the calpain digested JFP was estimated by electrophoresis and immunoblot analysis. METHODS Skinned fiber bundles were prepared from Skinned fibers and calpain treatment rabbit psoas muscle bundles (~2 mm wide) which had been treated with saponin (50 l@ml) for 60 min in a relaxing solution: (in mM) ATP, 4.76; MgMq, 5.54; KMs, 108.6; NaN3,20; PIPES, 20; pH 7.0. They were immersed in a rigor solution (KMs, 100, EGTA, 10; PIPES, 20; pH 7.0) before treatment with m-calpain (Sigma) in a digesting solution (KMs, 100; &MS;?, 2; PIPES, 20; pH 7.0). Fiber bundles were treated for 60 min or longer with 100 p.g calpain/ml usually at room temperature (22-24crJ). Skinned fiber bundles for the electrophoretic analysis contained 5-10 fibers and were -20 mm long. For the CICR measurements specimens contained a couple of fibers and were 2 mm in length. Measurement of CZCR rates A microfluorometric method (8) was used to measure CICR rates of the SR in skinned fibers mounted in a microcuvette. Since calpain cleaved regulatory proteins of the contractile system (9), calpain-treated skinned fibers went into irreversible contracture in the presence of MgATP even without C$+. Therefore, Ca2+ was passively loaded into the SR by reverse flux of Ca 2+ through CICR channels (10) for 60 s in the presence of 1 mM Ca2+ and 10 mM AMP. In calpain untreated fibers, the passively loaded caz” amounted about 35% of active loading in the presence of 4 mM MgATP at pCa 6.7 for 120 s. Amount of passive loading was the same when the loading duration was increased to 120 s, nor was dependent on the calpain treatment. After the Ca2+ loading both Ca2’ and AMP were washed away. Then as a test procedure, Ca2+ (buffered with 10 mM EGTA) was applied to induce CICR. C$+ and EGTA were then washed away and 40 pM fura- was introduced. 50 mM caffeine was then applied in the presence of fura- to thoroughly release remaining Ca2’ in the SR, of which amount was measured by the fluorescence intensity change at 500 nm with alternating excitation at 345 and 365 nm. The amount of Ca2+ released during the test procedure was estimated by comparing assays with and without the test procedure. Amount of Ca2+ in the SR declined almost exponentially to zero with increasing duration of the test procedure. Thus, the activity of CICR was expressed by the decay rate constant. Compositions of solutions and the further detail of the protocol are described elsewhere (8). Experiments were carried out at pH 7.0 and at room temperature. Electrophoresis and immunoblot analyses Proteins in skinned fibers were solubilized with 15 ~1 of solution containing 2% CHAPS, 2 mM EGTA, 0.1 mM (p-amidin phenyl) methane fluoride-HCl, 2 mM MgCl,, 200 mM NaCl, 20 mM potassium phosphate buffer (PH 7.0) for 60 min on ice. The extract was mixed with equal volume of a solution containing 5% SDS, 5% 2mercaptoethano1, and 0.1 M Tris-HCl buffer (pH 6.8), and 15 ~1 of the mixture was loaded onto each gel lane. Proteins were transferred from SDS polyacrylamide gels to nitrocellulose membrane (1 1), which was then treated with 5% non-fat milk before incubation for at least 60 min with a 714
Vol.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
polyclonal antibody against the synthetic decapeptide corresponding to the C-terminus end sequence of rabbit JFP (5, 6). The antibody (EC-3) was raised in guinea pig. The immunocomplexwasvisualized usingthe horseradishperoxidasereaction.
RESULTS Fig. 1 showsthe immunoblot analysisof calpain treated shinnedfibers. The mobility of JFP reacting with the anti-C-terminus antibody increasedafter 60 to 90 min treatment with mcalpain, and becamealmost the sameasthat of dystrophin (not shown). We have not so far found smallerproteolyic fragmentsreacting with the antibody even when we carried out more extensive treatment:increaseof the treatmenttime up to 360 min, three-fold increaseof calpainconcentration, treatmentat 3793,or useof thinner bundleswith only a coupleof fibers to facilitate diffusion of the enzyme. Fig. 2 showsthe ratesof CICR measuredat pCa 6.5,6.0 and 5.5 in skinned fibers with or without calpain treatment. The difference in the Ca2+ releaserate was small at the lowest Ca2+ concentration, whereas the CICR rates were increased by about 2-fold at the higher Ca2+ concentrations. We alsocarried out experimentsunderquasi-physiologicalcondition, i.e. in the presenceof Mg2+ and adenine nucleotide. To avoid both reuptake of C$+ into the SR and contraction of fibers we used2 mM AMPOPCP, a nonhydrolyzable ATP analog, in place of ATP. As shown in
RR.
6.5
6.0 PCs
Fig. 1. Immunoblot analysis of calpain digested JFP. Polyclonal antibody against the C-terminus sequence was used to stain JFP of calpain untreated (right lane) and treated skinned fibers. Calpain treatment was carried out for 60 min (left) or 90 min (center). R.R.: ryanodine nxeptor or JFP. Fig. 2. Effect of calpain treatment on the rates of CICR in the absence of both Mg2+ and nucleotide. Means and SEM. * 0.01 < P < 0.02, ** P c 0.01 (r-test). 715
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1.5 mM Mg, 2 mM AMPOPCP
F
.E a
Cl control E4 calpain
0.15
-
0.00
pCa 5.5
pCa 6.0
Fig. 3. Ratesof CICR in thepresence of 1.5mM Mg2+ and2 mM AMPOPCP with or without calpaintreatment. Numbersin the parentheses indicate the number of experiments. Means and SEM. ** P < 0.01.
Fig. 3 there was little effect of calpain treatmenton the rate of CICR at pCa 6, but 2-fold increase in the rate of Ca2+releasewas observedat pCa 5.5.
DISCUSSION The present study showsthat JFP is cleaved within muscle fibers by calpain to leave a polypeptide that includesthe C-terminus. Its mobility in SDS polyacrylamide gel was the sameas that of dystrophin whosemolecular massis -430 kDa (12). Since the molecularweight of JFP is -560 kDa (5,6), the cleavage site is located near residue 1200, and seemsto correspondto the protease-sensitiveregion I of JFP (13) (Fig. 4). This is probably one of the earliest proteolytic fragments of JFP in SR vesicle preparationsreported previously (7). In SR vesicles, JFP was further cleaved by calpain to about 150 kDa polypeptide. However, we have been so far unableto
Calpain clpvage site
I cl
* 1000 oorcine
N terminus domain Suppressor
I I II YY n
Surface oriented Im 2000
C terminus domain Ca channel Modulator sites
Fig. 4. Schematic diagram of the primary structure of JFP. Putative channel forming domains after two research groups (5,6) and surface oriented segments (12) are indicated, respectively, by the hatched bar and the boxes. The arrow indicates expected cleavage site of calpain. *Porcine malignant hyperthermia mutation is located at the 615th amino acid (14).
716
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cleave the 430 kDa fragment further. This may suggest that other cleavage sites are protected in skinned fibers. Even after most of JFP were cleaved by calpain, the CICR mechanism of the skinned fibers retained its Ca2’ sensitivity,
although the maximum rate of release was doubled in qualitative
agreement with the results obtained in SR vesicles incorporated
in lipid bilayers (7).
In the
previous study it was impossible to determine the molecular size of the incorporated JFP, and the correlation between the peptide length and the function was not decisive. We have now shown that -430 kDa peptide seems to contain Ca 2+ channel and Ca2+ binding sites as well as the C-terminus. Furthermore, AMPOPCP.
we found essentially
the same effect of calpain treatment in the presence of
Therefore, it is likely that the adenine nucleotide binding site is also located in the C-
terminus peptide. On the other hand the N-terminus peptide may suppress the activity of the CICR channels. In porcine malignant hyperthermia, the CICR is enhanced in a very similar manner as in the calpain treated samples (14). Such effect has been attributed to a point mutation that causes change of 615th amino acid from arginine to cysteine (15). This site is located in the middle of the N-terminus peptide of the present study. This seems to be consistent with the notion that the Nterminus domain is involved in the suppression of channel activity. The present results indicate that JFP is composed of functional domains. The N-terminus domain seems to modulate the opening of Ca2+ channel without nucleotide sensitivities.
changing Ca2+ or adenine
It will be interesting to see if that domain is involved in the excitation-
contraction coupling of skeletal muscle.
ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan and from the National Center for Neurology and Psychiatry of the Ministry of Health and Welfare of Japan.
REFERENCES 1. 2. 3. 4. 5.
Hymel, L.M., Inui, S., Fleischer, S. and Schindler, H. (1988) Proc. Nat. Acad. Sci. USA 85, 441445. Lai, F.A., Erickson, H.P., Rousseau, E., Liu, Q.-Y. and Meissner, G. (1988) Nature 331, 315-319. Imagawa, T., Smith, J.S., Coronado, R., Campbell, K.P. (1987) J. Biol. Chem. 262, 64606463. Endo, M. (1985) Curr. Top. Membr. Trunsp. 25, 181-230. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K. Minamino, N., Masuo, H., Ueda, M., Hanaoka, M., Hirose, T. and Numa, S. (1989) Narure 339, 439445. 717
Vol.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G. and MacLennan, D.H. (1990) J. Biol. Chem. 265,2244-2256. Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R. and Jones,L.R. (1990) Circ. Rex 67, 84-96. Iino, M. (1989) J. Gen. Physiol. 94, 363-383. Sugita, H., Ishiura, S., Suzuki, K. and Imahori, K. (1980) MuscleNerve 3, 335-339. Kitazawa, T. and Endo. M. (1976) Proc. JapanAcud 52,599~602. Reinnach, F.C., Masaki, T., Shafiq, T., Obinata, T. and Fishman, D.A. (1982) J. Cell. Biol. 95, 78-84. Koenig, M., Monaco, A.P. and Kunkel, L.M. (1988) Cell 53, 219-228. Marks, A.R., Fleischer, S. and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149. Ohta, T., Endo, M., Nakano, T., Morohoshi, Y., Wanikawa, K. and Ohga, A. (1989) Am . J. Physiol. 256, C358-C367. Fujii, J., Otsu, K., Zorzato, F., De Leon, S., Khanna, V.K., Weiler. J.E., O’Brien, P.J., MacLennan, D.H. (1991) Science253, 448-451.
718
