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Identification of a ∼170K subunit of the cardiac calcium channel using a monoclonal antibody to the skeletal muscle dihydropyridine receptor
Llfe Sciences, Vol. 43, pp. i055-i061 Printed in the U.S.A.
Pergamon Press
IDENTIFICATION OF A ~]7OK SUBUNIT OF THE CARDIAC CALCIUM CHANNEL USING A NONOCLONAL ANTIBODY TO THE SKELETAL MUSCLE DIHYDROPYRIDINE RECEPTOR Ren-Jie Chang and Henry Smtlowttz Department of Pharmacology. University of Connecticut Health Center, Farmtngton, CT 06032 (USA). (Received in final form August 8, 1988)
Su~ry Our newly Isolated monoclonal antibody (#78) s p e c t f f c a l l y i n t e r a c t s wlth the 170Kd 1,4 dthydropyrtdine binding component of the skeletal muscle calcium channel. Othydropyrtdtne receptor (OHPR) from rabbit skeletal muscle and canine cardiac membranes were p u r i f i e d by monoclonal antibody #78 a f f i n i t y chromatography. Ne show that DHPR from canine cardiac membranes l t k e DHPR from r a b b i t skeletal membranes contain a ~170Kd polypepttde to which antibody #78 tmmunoblots under both reductng and non-reducing conditions. In the l a s t three years a great deal of a t t e n t i o n has been focused on the voltage s e n s i t i v e ca|ctum channel. Several laboratories have attempted p u r i f i c a t i o n of the DHPR from both skeletal and cardiac muscle. Except f o r the data of Barhantn e t a ] . (1), basic agreement now exists that the complex that c o - p u r i f i e s wtth the DHP binding protein from skeletal muscle (2-7) consists of at least 170K, 140K, 55K and 30K pepttdes (reducing conditions) tn a 1:1:1:1 r a t i o (4). The polypepttde that migrates at 170K under both reducing and non-reducing conditions, contains the DHP binding s t t e (2) and ts d i s t i n c t from the polypepttde whose m o b i l i t y s h i f t s from 175K to 140K upon reduction. Because of the controversy that existed tn the skeletal muscle calcium channel f t e l d over the i d e n t i t y of the dthydropyrJdtne btndtng subuntt of the receptor, the behavior of t h i s protein under both reducing and non-reducing conditions has become an important criteria for tts Identification. The structure of the cardiac calctum channel c o w l e x ts less clear. Rengasamy et al (8) reported the enrichment of polypepttdes of 6OK. 54K and 34K tn t h e t r preparations. Schmtd et al. (9), reported the i d e n t i f i c a t i o n of a polypepttde of 170K under nonreduclng conditions, but t h e i r antiserum fatled to detect a htgh molecular weight component under reducing conditions. Cooper et al (10) and Takahasht and Cattera11 (11) i d e n t i f i e d polypepttdes of 170,000 daltons and 140,000 daltons before and a f t e r reduction respectively, wtth t h e i r polyclonal anttsera suggesting that e i t h e r the skeletal and cardiac calctum channels may d i f f e r wtth respect to the htgh molecular wetght subuntts or that they were tn f a c t looktng at the glycoprotetn whose mobtltty s h i f t s from 175K to 140K upon reduction. We have tsolated a monoclonal antibody t o the skeletal muscle DHPR (12). The anttbody tmmunopreclpttates skeletal muscle DHPR and tmmunoblots to a 170K polypepttde enriched tn skeletal T-tubule membranes which (a) does not s h i f t molecular weight upon reduction, (b) does not contain a detectab]e level of N-1tnked sugars, (c) ts phosphorylated by cycltc MIP protein ktnase, 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon P r e s s p l c
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(d) is photoafflnlty labeled by 3H-azldoptne as shown by 2D gel electrophorests and tmmunoblotting and (e) is completely d i s t i n c t from the 140K glycoprotein under reducing conditions as evidenced by 2D gel electrophorests and Immunoblottlng. These c r i t e r i a closely match those previously described f o r the 170Kd DHP binding subuntt of the DHPR of skeletal muscle (2-7). We have used antibody #78 to construct an a f f i n i t y column to p u r i f y the DHPR from both skeletal and cardiac membranes. Ne show in t h i s paper that membranes from dog heart contain a 170Kd polypepttde to which antibody #78 tmmunoblots under both reducing and non-reducing conditions. This is strong evidence that both the cardiac and skeletal calcium channels contain a large subuntt of ~170,000 apparent molecular weight with antigenic and structtonal s i m i l a r i t y . Methods Cardiac and Skeletal Muscle Membrane Preparations. Crude dog cardiac membranes were prepared by the method of Jones et e l . (13). Rabbit skeletal muscle heavy mtcrosomes were prepared by the method of Mitchell et e l . (14). Antibody |78 A f f i n i t y Column. 20mg Antibody #7B p u r i f i e d from culture supernatant by protein A (BiD Red) was linked to 1 ml A f f t g e l - l O (Bto Red) according to company instructions.
Purification of Skeletal and Cardiac DHPR by Antibody Affinity Chromatography. Membranes (35 mg) were extracted with I% dlgltonin in buffer A containing 25 mR HEPES, 185 mR KCI, l mR EDTA containing a protease inhibitor cocktail (l mR PMSF, 1 ~ Pepstatln A, l mg/ml antlpaln, 0.83 )M benzamldlne, 77 nM aprotlnln, l.l )M leupeptln). Detergent/protein ratio was I:5. Detergent extracts were clarified by ultracentrlfugatlon (160,000 xg for 40 mln) and incubated with 0.5 ml antibody linked Afflgel-lO overnight at 4°C with gentle rocking. The resin was packed onto a colum, washed with 20 ml Buffer A containing I% dlgltonln followed by 20 ml Buffer A containing 0.3% dlgltonin. DHPR were eluted with BloRad Elution Buffer (pH 3.0) containing 0.3% dlgltonln. Fractions (0.5 ml) were immediately neutralized with 65 )l IM Tris pH g.o. SDS Slab PAGE and Imnmnoblots. The method of Laemmlt (15) was used. Gradient slab (mini) gels were run at 150V f o r 1 hour. Samples were boiled f o r 3-5 minutes in 1% SOS with or without DTT (10 mR); iodoacetamide (25 mR) was added a f t e r b o t l i n g . Transfer to n i t r o c e l l u l o s e paper was done using a Hoefer Transphor u n i t f o r 2 hours. [mmunolabeling and peroxidase staining were done by published methods (Towbin et e l . ) (16). Results lmmunoprecipttatton of DHPR from Rabbit Skeletal and Dog Cardiac Membranes by Rab#78. Immunoprecipitatton was performed according to the method of Takahashi (11). Protein A p u r i f i e d NabS78 (llZg/lzl) gave maximal p r e c i p i t a t i o n of skeletal DHPR at a 1/100 di l u t t on (Figure 1, squares). No tmmunoprectpttatton was s e e n when Mouse IgG (llzg/lzl) was substituted f o r Nab#78 (Figure l , c i r c l e s ) or when receptors were pre-labeled with 3H-PN200-110 in the presence of a 400 fold molar excess of unlabeled PN
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Monoclonal Ab and Cardiac Ca 2+ Channels
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200-110 (Figure I , triangles). Maximal Immunopreclpitatlon was about 30% of the total ~H-PN200-110 labeled receptor present as determined by the FIG. l Mat~#78 immunoprecipitated OHPR. T-Tubule membranes (0.2 mg/ml in 50 mR Tris containing 32 pmol/mg 50001 protein DHP binding sites) were labeled with lOnM 4000F 3H-PN200-110 for 30 E 3000 minutes at 4% in the dark U in the absence ([]) (total labeling) and in the 2000 presence of 4 ~M unlabeled PN2OO-IIO(&) IO00 (non-specific labeling). The membranes were // L i J extracted for 30 minutes at 0 00001 0.001 001 4°C with digitonln (1% DILUTION OF ANTIBODY digitonin; detergent; protein = 5:1) and centrifuged at 160,000 xg for 40 minutes. Ten ~l of extract containing 0.3 pmoles of DHP receptor was incubated for 4 hours at 4°C with various dilutions of antibody #78 or normal mouse Ig6 (Sigma). Protein A sepharose reagent [made according to the method of Takahashi and Catterall, 1987 using rabbit antimouse Ig(G+A+M) serum (Zymed)] was incubated with each of the reaction mixtures overnight at 4°C. Complexes were collected by sedimentation (8,000 xg for 1.5 minutes) and washed twice in 0.5% digitonin in PBS. Pellets were dissolved in lO0 ~l 0.5% SOS overnight and assayed by s c i n t i l l a t i o n counting. A
PEG precipitation a s s a y after dlgltonln extraction. Since the immunoprecipitatlon assay takes about 15 hours and entails numerous dilutions, incubations and washes, some of the 3H-PN200-110 bound-label is inevitably lost due to ligand dissociation ( 4 , l l ) . Hence the 3H-PN200-1IO recovered in the immunoprecipitate is an underestimate of the labeled receptor in the detergent extract. Similar immunopreclpitation results w e r e obtained using cardiac membranes. Immunoprecipitatlon was performed as above (Method A) or using the Affigel-lO-Rab#78 a f f i n i t y resin described in Methods (Method B). Cardiac sarcolemmai membranes ~(33) (approximately 0.47pMoles 3H-PN200-1IO), werelabeled with aH-PN200-1IO and extracted with igitonin. Each assay tube contained 25fmoles digitonin extracted H-PN200-110 labeled DHPR and either a 1/50 dilution of Mab#78 (Method A) or 50~I of a f f i n i t y resin (Method B). Control assays were run in which the aH-PN200-110 pre-labeling was done in the p£esence of a large excess of unlabeled PN200-110. Eighty percent of the JH-PN200-110 bound cardiac DHPR was recovered on the a f f i n i t y resin compared to only 8% in the control reparatlon (Method B). Method A also gave specific precipitation of H-PN200-110 bound cardiac DHPR. Haxlmel Immunoprecipitation was 3 fold over control and represented 25% of the labeled receptor present in the assay. These results show that Nab#78 immunoprecipitates DHPR from both rabbit skeletal and dog cardiac membranes.
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Ca 2+
Channels
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Purification of OHPR from Rabbit Skeletal and 0oi Cardiac Men~)rances by Monoclonal Antibody Affinity ChromatoqraDhv. Thirty grams of rabbit skeletal muscle membranes (28 pMole DHP binding sites per n~j protein) were extracted with dlgltonln in the presence of protease inhibitor cocktail (Methods). The detergent extract was clarified by centrlfugatlon and incubated with Afflgel affinity resin overnight (4°C) wlth gentle rocking. The resin was then poured into a column, washed and OHPR was eluted wlth low pH buffer. Fractions were immediately neutralized and assayed for protein. Approximately 300 ~g of protein was recovered from the low pH eluates. Allquots (~g) were run on SOS PAGE under both non-reducing and reducing conditions. The Coomassle stained gel is shown in Figure 2A. Shown In lane a are molecular weight standards. Lanes b and c represent reduced and non-reduced conditions, respectively. Under non-reduced conditions, three main bands are evident at I80-1gOK, 170-1?SK and 62K. After reduction and alkylatlon, prominent bands are at 170-180K, 143-150K and 6OK. A minor band Is seen at 12OK. These bands have been previously observed for preparations of purified skeletal muscle DHPR (2-7). There was no material In the 30K region of the gel. The same procedure was followed using the canine cardiac membranes. T h i r t y grams of crude canine cardiac membranes (0.3 pmoles DHP bindlng sites per mg protein) were extracted with detergent, centrifuged, incubated wtth A f f t g e l a f f i n i t y resin and eluted as described above. As one might
FIG. 2 SOS PAGE of monoclonal antlbody afflnlty purified DHPR from A. Rabblt skeletal muscle membrane ( C o ~ s s l e staln) and 8. Canlne Cardiac Membranes (sllverstaln). Lane a, molecular weight markers; Lanes b and c, reduclng and nonreduclng condltlons, respectively. B. Canine cardiac ~ r a n e (S11ver staln). Lane b, molecular welght markers; lanes c and a, reducing and non-reducing conditions, respectlvely.
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Monoclonal Ab and Cardiac Ca 2+ Channels
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expect this preparation is far less pure since the starting material contained I/lO0 the concentration of OHPR. SOS PAGE and silver staining of this preparation revealed many bands (Figure 2B). While it is not possible to identify c ~ o n e n t s of the cardiac DHPR fro(, the silver stained gel, Immunoblots wlth antibody #78 positively identified a component of the cardiac calcium channel in this preparation. Monoclonal Antibody 178 Immunoblots to a I70K Protein in both Skeletal and Cardiac OHPR Preparations. no ~g low pH eluate from both rabbit skeletal muscle (Figure 3A lanes a,b) and canine cardiac muscle (Figure 3A lanes c,d) were run on SOS PAGE (mlnigel) under non-reducing (lanes a,c) and reducing (lanes b,d) conditions (see Methods). The proteins in the gel were electrophoresed onto nitrocellulose paper and assayed for menoclonal antibody binding (Methods). The Immunoblot revealed antibody binding to a band of ~I70K molecular weight which was present both in skeletal and cardiac membranes under both reducing and non-reducing conditions. The same result is shown in Figure 3B, another Immunoblot comparing skeletal (lane a) and cardiac (lane b) preparations run under reducing conditions. Previous attempts to reveal the Immunoblot using purified cardiac sarcolemmal membranes (~l pMole DHPR per mg protein) failed due to the low level of receptor present; partial purification of DHPR was required to visualize the blots.
•
B •
. ....
..,.
.~)01~,.~?~.~-.. ..
200k t TOk~
. : -...
.,~.
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cd
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....
o
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FIG. 3A,8 InTnunoblots with antibody #78 A. Lanes a and b, skeletal. Lanes c and d, cardiac. Lanes a and c, non-reduced. Lanes b and d, reduced. B. Lane a, skeletal (reduced). Lane b. cardiac (reduced). Discussion
The ability to identify the components of the cardiac OHPR has been mere difficult than the skeletal DHPR because there is far less DHPR in cardiac tissue. T-tubule preparations from skeletal muscle typically have 25-I00 pMoles OHPR per nxj membrane protein while purified cardiac sarcolemma contains 0.5-2 pMoles OHPR per nxj membrane protein. In this paper we have used a well characterized menoclonal antibody directed to the
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skeletal muscle DHPR (Figure 1,12) to show the presence of similar material in the cardiac preparation. At the concentrations of antibody employed in these experiments (I ~g/~l), monoclonal antibody #78 has not been observed to react s i g n i f i c a n t l y with any other skeletal microsomal protein other than the ITOK component of the DHPR purified by wheatgerm agglutinin chromatography (12). Hence the observed staining is very strong evidence that the cardiac membranes, l i k e the skeletal membrane, contain a 170K polypeptide component of the DHPRwhich does not s h i f t mobility on SOS PAGE after reduction. The minor band at 130-140K (Figure 2 A lane d) probably represents a proteolytic fragment of the 170 Kd protein which retains the antibody recognition site and is augmented by repeated freezing and thawing of the sample. We suggest that the previous report by Rengasamy et al. (8) was incorrect since they failed to purify a high molecular weight polypeptide. Further Schmid et al. (g), Cooper et al. (lO) and Takahashi and Catterall ( l l ) failed to detect the 170K polypeptide under reducing conditions probably due to either the proteolytic conversion of 170K polypeptide to a 140K polypeptide revealed under reducing conditions or to the s p e c i f i c i t y of t h e i r antisera. Because of the controversy that existed in the skeletal calcium channel f i e l d over the i d e n t i t y of the h l g h molecular weight subunit of the dihydropyridine receptor, the issue of the effect of reducing conditions on the apparent molecular weight of the cardiac protein is a significant one. Enrichment of a 170K polypeptide in the purified DHPR preparations of luana et al. (17) is d i f f i c u l t to discern from the data andthere is no comparison of t h e i r preparation under both reducing and non-reducing conditions. Our data provides the f i r s t strong evidence that the cardiac DHPR and the skeletal DHPR both contain a very similar polypeptide of approximately 170K molecular weight that shares a common antibody binding site and does not change mobility on SDS PAGE upon reduction. Acknowledgments This work was supported by the Program Project grant HL33026. I. 2. 3. 4. 5. 6. 7. 8. 9. lO. ll. 12.
REFERENCES 3. BARHANIN, 3. COPPOLA, A. SCHMID, M. BORSOITO and M. LAZDUNSKI, Eur. 3. Biochem. 164, 525-531 (1987). A . T . LEUNG, T. IMAGAWA, and K.P. CAMPBELL, O. Biol. Chem. 262, 7943-7946 (1987). A.H. SHARP, 1. IMAGAWA, A . T . LEUNG and K.P. CAMPBELL, Biol. Chem. 262, 12309-12315 (2987). N. TAKAHASHI, M.3. SEA6AR, 3.F. JONES, B.F.X. REBER and W.A. CATTERALL, Proc. Natl. Acad. Sci. 84, 5478-5482 (1987). P.L. VAGHY, 3. STRIESSNIG, M. KUNIHISA, H-G. KRAVS, K. ]TAGAKI, E. McKENNA, H. GLOSSMANN and A. SCHWARIZ, 3. B i o l . Chem. 262, 14337-14342 (1987). w. NAISTANZYK, A. R~HRKASIEN, M. SIEBER, C. RUDOLPH, C. SCH~CHIELE, D. MARNE AND F. HOFMANN, Eur. 3. Biochem. 1§9, 137-142 (1987). M.E. MORTONand S.C. FRUEHNER, J. B i o l . Chem. 262, 11904-11907 (1987). A. RENGASAMY, J. PTASIENSKI and M.M. HOSEY, Biochem. Biophys. Res. Commun. 126, I-7 (1985). A. SCHMID, J. BARHANIN, T. COPPULA, M. BORSOTIO and M. LAZDUNSKI, Biochem. 25, 3492-3495 (1986). C . L . COOPER, S. VANDAELE, J. BARHANIN, M. FOSSET, M. LAZOUNSKI and M.N. HOSEY, 3. Biol. Chem. 262, 509-512 (1987). M. TAKAHASHI and W.A. CATIERALL, Science 236, 8B-91 (1987). H. SMILOWllZ, R - J . CHANG and C. BOWIK, Soc. for Neuroscience Abst. (1987).
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13. 14. 15. 16. 17.
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L.R. JONES, S.W. HADDOCK, O.R. BESCH J r . , J. Biol. Chem. 255, 9971-9980 (1980). R.D. MTTCHELL, P. PALADE and S. FLETSHER, J. Cell Biol. 96, 1008-1016 (1983). U.K. LAEI414LI, Nature 227, 680-685 (1970). H. TOWB[N, J. STAEHELTN and 3. GORDON, Proc. Natl. Acad. Sct. 76, 4350-4354 (1979). B.S. TUANA, B.J. HURPHY and Q. YT, Holec. and Cell gtochem. 76, 173-184 (1987).
Note added in proof: After this manuscript was submitted, Schneider, T. and Hofmann, F. Eur. J. Biochem. 124, 369-375, 1988, appeared suggesting that the ~i component of the cardiac L-type DHPR has an apparent molecular weight that is about 10% larger than its skeletal counterpart. Our data shows that the dog cardiac ~i appears slightly larger than the rabbit skeletal ~i; however additional work is needed to further analyze and compare the cardiac and skeletal DHPR.
Pergamon Press
IDENTIFICATION OF A ~]7OK SUBUNIT OF THE CARDIAC CALCIUM CHANNEL USING A NONOCLONAL ANTIBODY TO THE SKELETAL MUSCLE DIHYDROPYRIDINE RECEPTOR Ren-Jie Chang and Henry Smtlowttz Department of Pharmacology. University of Connecticut Health Center, Farmtngton, CT 06032 (USA). (Received in final form August 8, 1988)
Su~ry Our newly Isolated monoclonal antibody (#78) s p e c t f f c a l l y i n t e r a c t s wlth the 170Kd 1,4 dthydropyrtdine binding component of the skeletal muscle calcium channel. Othydropyrtdtne receptor (OHPR) from rabbit skeletal muscle and canine cardiac membranes were p u r i f i e d by monoclonal antibody #78 a f f i n i t y chromatography. Ne show that DHPR from canine cardiac membranes l t k e DHPR from r a b b i t skeletal membranes contain a ~170Kd polypepttde to which antibody #78 tmmunoblots under both reductng and non-reducing conditions. In the l a s t three years a great deal of a t t e n t i o n has been focused on the voltage s e n s i t i v e ca|ctum channel. Several laboratories have attempted p u r i f i c a t i o n of the DHPR from both skeletal and cardiac muscle. Except f o r the data of Barhantn e t a ] . (1), basic agreement now exists that the complex that c o - p u r i f i e s wtth the DHP binding protein from skeletal muscle (2-7) consists of at least 170K, 140K, 55K and 30K pepttdes (reducing conditions) tn a 1:1:1:1 r a t i o (4). The polypepttde that migrates at 170K under both reducing and non-reducing conditions, contains the DHP binding s t t e (2) and ts d i s t i n c t from the polypepttde whose m o b i l i t y s h i f t s from 175K to 140K upon reduction. Because of the controversy that existed tn the skeletal muscle calcium channel f t e l d over the i d e n t i t y of the dthydropyrJdtne btndtng subuntt of the receptor, the behavior of t h i s protein under both reducing and non-reducing conditions has become an important criteria for tts Identification. The structure of the cardiac calctum channel c o w l e x ts less clear. Rengasamy et al (8) reported the enrichment of polypepttdes of 6OK. 54K and 34K tn t h e t r preparations. Schmtd et al. (9), reported the i d e n t i f i c a t i o n of a polypepttde of 170K under nonreduclng conditions, but t h e i r antiserum fatled to detect a htgh molecular weight component under reducing conditions. Cooper et al (10) and Takahasht and Cattera11 (11) i d e n t i f i e d polypepttdes of 170,000 daltons and 140,000 daltons before and a f t e r reduction respectively, wtth t h e i r polyclonal anttsera suggesting that e i t h e r the skeletal and cardiac calctum channels may d i f f e r wtth respect to the htgh molecular wetght subuntts or that they were tn f a c t looktng at the glycoprotetn whose mobtltty s h i f t s from 175K to 140K upon reduction. We have tsolated a monoclonal antibody t o the skeletal muscle DHPR (12). The anttbody tmmunopreclpttates skeletal muscle DHPR and tmmunoblots to a 170K polypepttde enriched tn skeletal T-tubule membranes which (a) does not s h i f t molecular weight upon reduction, (b) does not contain a detectab]e level of N-1tnked sugars, (c) ts phosphorylated by cycltc MIP protein ktnase, 0024-3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon P r e s s p l c
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(d) is photoafflnlty labeled by 3H-azldoptne as shown by 2D gel electrophorests and tmmunoblotting and (e) is completely d i s t i n c t from the 140K glycoprotein under reducing conditions as evidenced by 2D gel electrophorests and Immunoblottlng. These c r i t e r i a closely match those previously described f o r the 170Kd DHP binding subuntt of the DHPR of skeletal muscle (2-7). We have used antibody #78 to construct an a f f i n i t y column to p u r i f y the DHPR from both skeletal and cardiac membranes. Ne show in t h i s paper that membranes from dog heart contain a 170Kd polypepttde to which antibody #78 tmmunoblots under both reducing and non-reducing conditions. This is strong evidence that both the cardiac and skeletal calcium channels contain a large subuntt of ~170,000 apparent molecular weight with antigenic and structtonal s i m i l a r i t y . Methods Cardiac and Skeletal Muscle Membrane Preparations. Crude dog cardiac membranes were prepared by the method of Jones et e l . (13). Rabbit skeletal muscle heavy mtcrosomes were prepared by the method of Mitchell et e l . (14). Antibody |78 A f f i n i t y Column. 20mg Antibody #7B p u r i f i e d from culture supernatant by protein A (BiD Red) was linked to 1 ml A f f t g e l - l O (Bto Red) according to company instructions.
Purification of Skeletal and Cardiac DHPR by Antibody Affinity Chromatography. Membranes (35 mg) were extracted with I% dlgltonin in buffer A containing 25 mR HEPES, 185 mR KCI, l mR EDTA containing a protease inhibitor cocktail (l mR PMSF, 1 ~ Pepstatln A, l mg/ml antlpaln, 0.83 )M benzamldlne, 77 nM aprotlnln, l.l )M leupeptln). Detergent/protein ratio was I:5. Detergent extracts were clarified by ultracentrlfugatlon (160,000 xg for 40 mln) and incubated with 0.5 ml antibody linked Afflgel-lO overnight at 4°C with gentle rocking. The resin was packed onto a colum, washed with 20 ml Buffer A containing I% dlgltonln followed by 20 ml Buffer A containing 0.3% dlgltonin. DHPR were eluted with BloRad Elution Buffer (pH 3.0) containing 0.3% dlgltonln. Fractions (0.5 ml) were immediately neutralized with 65 )l IM Tris pH g.o. SDS Slab PAGE and Imnmnoblots. The method of Laemmlt (15) was used. Gradient slab (mini) gels were run at 150V f o r 1 hour. Samples were boiled f o r 3-5 minutes in 1% SOS with or without DTT (10 mR); iodoacetamide (25 mR) was added a f t e r b o t l i n g . Transfer to n i t r o c e l l u l o s e paper was done using a Hoefer Transphor u n i t f o r 2 hours. [mmunolabeling and peroxidase staining were done by published methods (Towbin et e l . ) (16). Results lmmunoprecipttatton of DHPR from Rabbit Skeletal and Dog Cardiac Membranes by Rab#78. Immunoprecipitatton was performed according to the method of Takahashi (11). Protein A p u r i f i e d NabS78 (llZg/lzl) gave maximal p r e c i p i t a t i o n of skeletal DHPR at a 1/100 di l u t t on (Figure 1, squares). No tmmunoprectpttatton was s e e n when Mouse IgG (llzg/lzl) was substituted f o r Nab#78 (Figure l , c i r c l e s ) or when receptors were pre-labeled with 3H-PN200-110 in the presence of a 400 fold molar excess of unlabeled PN
Vol. 43, No. 13, 1988
Monoclonal Ab and Cardiac Ca 2+ Channels
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200-110 (Figure I , triangles). Maximal Immunopreclpitatlon was about 30% of the total ~H-PN200-110 labeled receptor present as determined by the FIG. l Mat~#78 immunoprecipitated OHPR. T-Tubule membranes (0.2 mg/ml in 50 mR Tris containing 32 pmol/mg 50001 protein DHP binding sites) were labeled with lOnM 4000F 3H-PN200-110 for 30 E 3000 minutes at 4% in the dark U in the absence ([]) (total labeling) and in the 2000 presence of 4 ~M unlabeled PN2OO-IIO(&) IO00 (non-specific labeling). The membranes were // L i J extracted for 30 minutes at 0 00001 0.001 001 4°C with digitonln (1% DILUTION OF ANTIBODY digitonin; detergent; protein = 5:1) and centrifuged at 160,000 xg for 40 minutes. Ten ~l of extract containing 0.3 pmoles of DHP receptor was incubated for 4 hours at 4°C with various dilutions of antibody #78 or normal mouse Ig6 (Sigma). Protein A sepharose reagent [made according to the method of Takahashi and Catterall, 1987 using rabbit antimouse Ig(G+A+M) serum (Zymed)] was incubated with each of the reaction mixtures overnight at 4°C. Complexes were collected by sedimentation (8,000 xg for 1.5 minutes) and washed twice in 0.5% digitonin in PBS. Pellets were dissolved in lO0 ~l 0.5% SOS overnight and assayed by s c i n t i l l a t i o n counting. A
PEG precipitation a s s a y after dlgltonln extraction. Since the immunoprecipitatlon assay takes about 15 hours and entails numerous dilutions, incubations and washes, some of the 3H-PN200-110 bound-label is inevitably lost due to ligand dissociation ( 4 , l l ) . Hence the 3H-PN200-1IO recovered in the immunoprecipitate is an underestimate of the labeled receptor in the detergent extract. Similar immunopreclpitation results w e r e obtained using cardiac membranes. Immunoprecipitatlon was performed as above (Method A) or using the Affigel-lO-Rab#78 a f f i n i t y resin described in Methods (Method B). Cardiac sarcolemmai membranes ~(33) (approximately 0.47pMoles 3H-PN200-1IO), werelabeled with aH-PN200-1IO and extracted with igitonin. Each assay tube contained 25fmoles digitonin extracted H-PN200-110 labeled DHPR and either a 1/50 dilution of Mab#78 (Method A) or 50~I of a f f i n i t y resin (Method B). Control assays were run in which the aH-PN200-110 pre-labeling was done in the p£esence of a large excess of unlabeled PN200-110. Eighty percent of the JH-PN200-110 bound cardiac DHPR was recovered on the a f f i n i t y resin compared to only 8% in the control reparatlon (Method B). Method A also gave specific precipitation of H-PN200-110 bound cardiac DHPR. Haxlmel Immunoprecipitation was 3 fold over control and represented 25% of the labeled receptor present in the assay. These results show that Nab#78 immunoprecipitates DHPR from both rabbit skeletal and dog cardiac membranes.
~
~
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Monoclonal Ab and Cardiac
Ca 2+
Channels
Vol. 43, No. 13, 1988
Purification of OHPR from Rabbit Skeletal and 0oi Cardiac Men~)rances by Monoclonal Antibody Affinity ChromatoqraDhv. Thirty grams of rabbit skeletal muscle membranes (28 pMole DHP binding sites per n~j protein) were extracted with dlgltonln in the presence of protease inhibitor cocktail (Methods). The detergent extract was clarified by centrlfugatlon and incubated with Afflgel affinity resin overnight (4°C) wlth gentle rocking. The resin was then poured into a column, washed and OHPR was eluted wlth low pH buffer. Fractions were immediately neutralized and assayed for protein. Approximately 300 ~g of protein was recovered from the low pH eluates. Allquots (~g) were run on SOS PAGE under both non-reducing and reducing conditions. The Coomassle stained gel is shown in Figure 2A. Shown In lane a are molecular weight standards. Lanes b and c represent reduced and non-reduced conditions, respectively. Under non-reduced conditions, three main bands are evident at I80-1gOK, 170-1?SK and 62K. After reduction and alkylatlon, prominent bands are at 170-180K, 143-150K and 6OK. A minor band Is seen at 12OK. These bands have been previously observed for preparations of purified skeletal muscle DHPR (2-7). There was no material In the 30K region of the gel. The same procedure was followed using the canine cardiac membranes. T h i r t y grams of crude canine cardiac membranes (0.3 pmoles DHP bindlng sites per mg protein) were extracted with detergent, centrifuged, incubated wtth A f f t g e l a f f i n i t y resin and eluted as described above. As one might
FIG. 2 SOS PAGE of monoclonal antlbody afflnlty purified DHPR from A. Rabblt skeletal muscle membrane ( C o ~ s s l e staln) and 8. Canlne Cardiac Membranes (sllverstaln). Lane a, molecular weight markers; Lanes b and c, reduclng and nonreduclng condltlons, respectively. B. Canine cardiac ~ r a n e (S11ver staln). Lane b, molecular welght markers; lanes c and a, reducing and non-reducing conditions, respectlvely.
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Monoclonal Ab and Cardiac Ca 2+ Channels
1059
expect this preparation is far less pure since the starting material contained I/lO0 the concentration of OHPR. SOS PAGE and silver staining of this preparation revealed many bands (Figure 2B). While it is not possible to identify c ~ o n e n t s of the cardiac DHPR fro(, the silver stained gel, Immunoblots wlth antibody #78 positively identified a component of the cardiac calcium channel in this preparation. Monoclonal Antibody 178 Immunoblots to a I70K Protein in both Skeletal and Cardiac OHPR Preparations. no ~g low pH eluate from both rabbit skeletal muscle (Figure 3A lanes a,b) and canine cardiac muscle (Figure 3A lanes c,d) were run on SOS PAGE (mlnigel) under non-reducing (lanes a,c) and reducing (lanes b,d) conditions (see Methods). The proteins in the gel were electrophoresed onto nitrocellulose paper and assayed for menoclonal antibody binding (Methods). The Immunoblot revealed antibody binding to a band of ~I70K molecular weight which was present both in skeletal and cardiac membranes under both reducing and non-reducing conditions. The same result is shown in Figure 3B, another Immunoblot comparing skeletal (lane a) and cardiac (lane b) preparations run under reducing conditions. Previous attempts to reveal the Immunoblot using purified cardiac sarcolemmal membranes (~l pMole DHPR per mg protein) failed due to the low level of receptor present; partial purification of DHPR was required to visualize the blots.
•
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200k t TOk~
. : -...
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cd
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....
o
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FIG. 3A,8 InTnunoblots with antibody #78 A. Lanes a and b, skeletal. Lanes c and d, cardiac. Lanes a and c, non-reduced. Lanes b and d, reduced. B. Lane a, skeletal (reduced). Lane b. cardiac (reduced). Discussion
The ability to identify the components of the cardiac OHPR has been mere difficult than the skeletal DHPR because there is far less DHPR in cardiac tissue. T-tubule preparations from skeletal muscle typically have 25-I00 pMoles OHPR per nxj membrane protein while purified cardiac sarcolemma contains 0.5-2 pMoles OHPR per nxj membrane protein. In this paper we have used a well characterized menoclonal antibody directed to the
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Monoclonal Ab and Cardiac Ca 2+ Channels
Vol. 43, No. 13, 1988
skeletal muscle DHPR (Figure 1,12) to show the presence of similar material in the cardiac preparation. At the concentrations of antibody employed in these experiments (I ~g/~l), monoclonal antibody #78 has not been observed to react s i g n i f i c a n t l y with any other skeletal microsomal protein other than the ITOK component of the DHPR purified by wheatgerm agglutinin chromatography (12). Hence the observed staining is very strong evidence that the cardiac membranes, l i k e the skeletal membrane, contain a 170K polypeptide component of the DHPRwhich does not s h i f t mobility on SOS PAGE after reduction. The minor band at 130-140K (Figure 2 A lane d) probably represents a proteolytic fragment of the 170 Kd protein which retains the antibody recognition site and is augmented by repeated freezing and thawing of the sample. We suggest that the previous report by Rengasamy et al. (8) was incorrect since they failed to purify a high molecular weight polypeptide. Further Schmid et al. (g), Cooper et al. (lO) and Takahashi and Catterall ( l l ) failed to detect the 170K polypeptide under reducing conditions probably due to either the proteolytic conversion of 170K polypeptide to a 140K polypeptide revealed under reducing conditions or to the s p e c i f i c i t y of t h e i r antisera. Because of the controversy that existed in the skeletal calcium channel f i e l d over the i d e n t i t y of the h l g h molecular weight subunit of the dihydropyridine receptor, the issue of the effect of reducing conditions on the apparent molecular weight of the cardiac protein is a significant one. Enrichment of a 170K polypeptide in the purified DHPR preparations of luana et al. (17) is d i f f i c u l t to discern from the data andthere is no comparison of t h e i r preparation under both reducing and non-reducing conditions. Our data provides the f i r s t strong evidence that the cardiac DHPR and the skeletal DHPR both contain a very similar polypeptide of approximately 170K molecular weight that shares a common antibody binding site and does not change mobility on SDS PAGE upon reduction. Acknowledgments This work was supported by the Program Project grant HL33026. I. 2. 3. 4. 5. 6. 7. 8. 9. lO. ll. 12.
REFERENCES 3. BARHANIN, 3. COPPOLA, A. SCHMID, M. BORSOITO and M. LAZDUNSKI, Eur. 3. Biochem. 164, 525-531 (1987). A . T . LEUNG, T. IMAGAWA, and K.P. CAMPBELL, O. Biol. Chem. 262, 7943-7946 (1987). A.H. SHARP, 1. IMAGAWA, A . T . LEUNG and K.P. CAMPBELL, Biol. Chem. 262, 12309-12315 (2987). N. TAKAHASHI, M.3. SEA6AR, 3.F. JONES, B.F.X. REBER and W.A. CATTERALL, Proc. Natl. Acad. Sci. 84, 5478-5482 (1987). P.L. VAGHY, 3. STRIESSNIG, M. KUNIHISA, H-G. KRAVS, K. ]TAGAKI, E. McKENNA, H. GLOSSMANN and A. SCHWARIZ, 3. B i o l . Chem. 262, 14337-14342 (1987). w. NAISTANZYK, A. R~HRKASIEN, M. SIEBER, C. RUDOLPH, C. SCH~CHIELE, D. MARNE AND F. HOFMANN, Eur. 3. Biochem. 1§9, 137-142 (1987). M.E. MORTONand S.C. FRUEHNER, J. B i o l . Chem. 262, 11904-11907 (1987). A. RENGASAMY, J. PTASIENSKI and M.M. HOSEY, Biochem. Biophys. Res. Commun. 126, I-7 (1985). A. SCHMID, J. BARHANIN, T. COPPULA, M. BORSOTIO and M. LAZDUNSKI, Biochem. 25, 3492-3495 (1986). C . L . COOPER, S. VANDAELE, J. BARHANIN, M. FOSSET, M. LAZOUNSKI and M.N. HOSEY, 3. Biol. Chem. 262, 509-512 (1987). M. TAKAHASHI and W.A. CATIERALL, Science 236, 8B-91 (1987). H. SMILOWllZ, R - J . CHANG and C. BOWIK, Soc. for Neuroscience Abst. (1987).
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13. 14. 15. 16. 17.
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L.R. JONES, S.W. HADDOCK, O.R. BESCH J r . , J. Biol. Chem. 255, 9971-9980 (1980). R.D. MTTCHELL, P. PALADE and S. FLETSHER, J. Cell Biol. 96, 1008-1016 (1983). U.K. LAEI414LI, Nature 227, 680-685 (1970). H. TOWB[N, J. STAEHELTN and 3. GORDON, Proc. Natl. Acad. Sct. 76, 4350-4354 (1979). B.S. TUANA, B.J. HURPHY and Q. YT, Holec. and Cell gtochem. 76, 173-184 (1987).
Note added in proof: After this manuscript was submitted, Schneider, T. and Hofmann, F. Eur. J. Biochem. 124, 369-375, 1988, appeared suggesting that the ~i component of the cardiac L-type DHPR has an apparent molecular weight that is about 10% larger than its skeletal counterpart. Our data shows that the dog cardiac ~i appears slightly larger than the rabbit skeletal ~i; however additional work is needed to further analyze and compare the cardiac and skeletal DHPR.