Oligomeric Status of the Dihydropyridine Receptor in Aged Skeletal Muscle

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Molecular Cell Biology Research Communications 4, 224–229 (2000)

doi:10.1006/mcbr.2001.0282, available online at http://www.idealibrary.com on

Oligomeric Status of the Dihydropyridine Receptorin Aged Skeletal Muscle

Michelle Ryan,* Bruce M. Carlson,† and Kay Ohlendieck*,1

*Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin,Belfield, Dublin 4, Ireland; and †Department of Anatomy and Cell Biology, University of Michigan,Ann Arbor, Michigan 48109-1166

Received March 1, 2001; published online May 24, 2001

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A prominent feature of aging is represented by a de-crease in muscle mass and strength. Abnormalities inCa21-regulatory membrane complexes are involved in

any muscular disorders. In analogy, we determinedotential age-related changes in a key component ofxcitation-contraction coupling, the dihydropyridine re-eptor. Immunoblotting of the microsomal fraction fromged rabbit muscle revealed a drastic decline in theoltage-sensing a1-subunit of this transverse-tubular re-

ceptor, but only marginally altered expression of its aux-iliary a2-subunit and the Na1/K1-ATPase. A shift toslower fibre type characteristics was indicated by anage-related increase in the slow calsequestrin isoform.Chemical crosslinking analysis showed that the triadreceptor complex has a comparable tendency of protein–protein interactions in young and aged muscles. Hence,a reduced expression and not modified oligomerizationof the principal dihydropyridine receptor subunit mightbe involved in triggering impaired triadic signal trans-duction and abnormal Ca21-homeostasis resulting in aprogressive functional decline of skeletal muscles. © 2001

Academic Press

Aging is a fundamental biological process and sincethe senescent organism is predisposed to a variety ofpathologies, gerontology research has gained great in-terest in the biomedical field (1). In order to both eval-uate age-related changes and to develop strategies tocounteract its deleterious effects on cellular function,an understanding of the molecular changes occurringduring aging is essential (2). Skeletal muscle repre-sents the single most abundant tissue in mammals andwith the advancement of age, muscle fibres undergomany structural and functional changes (3). Prominentbiochemical, cell biological, and physiological featuresof this decline are an overall loss of muscle mass due to

1 To whom correspondence should be addressed. Fax: (353) (1)269-2749. E-mail: [email protected].

2241522-4724/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

muscle fibre atrophy and a loss of myogenic and ma-ture cells, a shift to slower fibre types, loss of motorneurons/units, a reduced ability of satellite cells tointegrate into muscle fibres, as well as abnormal me-tabolism, bioenergetics, and ion homeostasis (4–7) Theexact molecular mechanisms underlying these alter-ations are still elusive, although it is clear that numer-ous molecular and cellular factors may influence therepair and adaptation of aging muscle fibres (8). Onepossible pathophysiological scenario is that alterationsin the capacity to maintain normal Ca21-homeostasisare involved in the aging processes (9, 10). We there-fore investigated in this study potential age-relatedchanges in a key component of excitation-contractioncoupling, the dihydropyridine receptor.

Excitation-contraction coupling at the triadic junc-tion between the transverse tubular membrane systemand the sarcoplasmic reticulum represents a uniquesignal transduction event (11). Depolarisation of themuscle surface membrane leads to Ca21-release fromthe lumen of the terminal cisternae. In mature skeletalmuscle fibres, signal transduction is mediated by adirect physical linkage between the voltage-sensinga1-subunit of the dihydropyridine receptor and the ry-anodine receptor Ca21-release channel (12). In con-rast, in developing skeletal muscle a cardiac-like modef excitation-contraction coupling exists (13). Duringarly myogenesis, Ca21-ions initially enter the cytosol

via activated surface dihydropyridine receptors, whichthen triggers a Ca21-induced Ca21-release mechanismvia ryanodine receptor complexes (14). The dihydropyr-idine class of receptors consists of the principal func-tional a1-subunit of apparent 175 kDa which forms theL-type Ca21-channel pore and represents the voltage-sensing unit of the muscle membrane periphery, aswell as four additional subunits, the a2/d-subunit com-

lex of 143/27 kDa, the b-subunit of 54 kDa, and theg-subunit of apparent 30 kDa (15). The auxiliary sub-units were shown to play an essential role in the propertargeting of the a1-subunit to the surface membrane

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Vol. 4, No. 4, 2000 MOLECULAR CELL BIOLOGY RESEARCH COMMUNICATIONS

and to enhance the ability of the principal receptorsubunit to sense depolarisations or to act as a Ca21-channel (16).

Different motorneuron activity has a profound influ-ence on the levels and pattern of isoform expression ofCa21-regulatory membrane proteins including the di-

ydropyridine receptor (17). During fibre type shiftings experimentally induced by chronic electrostimula-ion, an exchange between the a1S-subunit and the

a1C-subunit isoforms of the voltage sensor occurs (18).These molecular changes in a key Ca21-handling pro-tein reflect adaptations to changed physiological de-mands and show that protein expression in skeletalmuscle fibres is strongly dependent on external factors(19). In analogy, age-related changes in the innerva-tion pattern of skeletal muscles may contribute to vari-ations in the relative density and isoform expression ofmuscle-specific components. We therefore performedan immunoblot and chemical crosslinking analysis inorder to determine potential alterations in a key com-ponent of excitation-contraction coupling, the dihydro-pyridine receptor, during aging.

MATERIALS AND METHODS

Materials. Acrylamide stock solutions, protease in-hibitors and peroxidase-conjugated secondary antibod-ies were purchased from Boehringer Mannheim(Lewis, East Sussex, UK). Monoclonal antibody (mAb)IIID5 against the a1-subunit of the dihydropyridinereceptor was a generous gift from Kevin P. Campbell(Howard Hughes Medical Institute, University of Iowa,Iowa City, IA). Commercially available primary anti-bodies were from Upstate Biotechnology (Lake Placid,NY) (mAb c464.6 to the Na1/K1-ATPase) and AffinityBioreagents (Golden, CO) (mAb 20A to the a2-subunitof the dihydropyridine receptor; mAb VIIID12 to fastcalsequestrin, pAb to slow calsequestrin). Westernblotting chemiluminescence substrates and the chem-ical crosslinker dithiobis-succinimidyl-propionate wereobtained from Pierce & Warriner (Chester, Cheshire,UK). Immobilon-P nitrocellulose was from MilliporeCorporation (Bedford, MA). All other chemicals were ofanalytical grade and purchased from Sigma ChemicalCompany (Poole, Dorset, UK).

Isolation of muscle membranes. Muscle samplesfrom aging male New Zealand white rabbits (3 weeksto 2.4 years of age) and male WI/HicksCar rats (4 and28 months of age) were obtained from the biomedicalfacilities of University College Dublin and the Univer-sity of Michigan at Ann Arbor, respectively. For immu-noblot analysis, total skeletal muscle specimens weredissected and quick-frozen in liquid nitrogen-cooledisopentane, transported on dry ice and stored at 270°Cprior to usage. In order to directly compare the relativeexpression levels of the dihydropyridine receptor andother membrane proteins in differently aged skeletal

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muscle fibres, established protocols for the isolation ofmicrosomal membranes from muscle homogenateswere employed (20). To minimize proteolytic degrada-tion of muscle membrane proteins, all buffers weresupplemented with a protease inhibitor cocktail (0.2mM pefabloc, 1.4 mM pepstatin, 0.15 mM aprotinin, 0.3mM E-64, 1 mM leupeptin, 0.5 mM soybean trypsininhibitor, and 1mM EDTA) and all procedures wereperformed in a cold room at 0–4°C. Membrane pelletswere resuspended at a protein concentration of 10mg/ml and used immediately for gel electrophoreticanalysis or quick-frozen in liquid nitrogen and thenstored at 270°C prior to further usage. Protein concen-ration was determined by the method of Bradford (21)sing bovine serum albumin as a standard.

Chemical crosslinking analysis. Chemical crosslink-ng was performed as previously described in detail13, 20). Microsomes (1 mg protein) were diluted to anal volume of 500 ml with 50 mM Hepes, pH 8.0 at

25°C. Using a stock solution of 5 mg/ml chemicalcrosslinker, dithio-bis-succinimidyl-propionate (DSP)was added to the membrane suspension at a final con-centration of 0 to 25 mg cross-linker per mg membraneprotein. Samples were incubated for 30 min with con-stant agitation at 25°C and then the crosslinking reac-tions terminated by the addition of 50 ml of 1 M am-monium acetate per ml reaction mixture. An equalvolume of reducing sample buffer (22) was added andthe solution incubated for 10 min at 37°C before beingsubjected to electrophoretic separation.

Gel electrophoresis and immunoblotting. Gel elec-trophoretic separation using 7% (w/v) resolving gelswith a 5% (w/v) stacking gel in the presence of sodiumdodecyl sulfate and dithiotreitol was performed for 280Vh employing a Mini-MP3 electrophoresis system fromBio-Rad Laboratories (Hempel Hempstead, Herts.,UK), whereby 30 mg protein was loaded per well (23).Chemically crosslinked samples were separated on 7%(w/v) resolving gels lacking a stacking gel system. Ni-trocellulose replica of polyacrylamide gels were pro-duced as described by Towbin et al. (24). For immuno-labeling, nitrocellulose sheets were blocked andincubated with primary and secondary antibodies aspreviously described (20). Immunodecoration was eval-uated by the enhanced chemiluminescence technique(25). Densitometric scanning of enhanced chemilumi-nescence blots was performed on a Molecular Dynam-ics 300S computing densitometer (Sunnyvale, CA) withImageQuant V3.0 software.

RESULTS AND DISCUSSION

Primary or secondary changes in Ca21-regulationhave previously been described to be the underlyingcause for many different muscle pathologies (26, 27).Susceptibility to the drug-induced pharmacogenetic

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muscle disorder malignant hyperthermia is based onmutations in the Ca21-release channel or the L-typeCa21-channel of the dihydropyridine receptor class (28,

FIG. 1. Immunoblot analysis of the dihydropyridine receptor in a(a, rabbit; g, rat) and identical immunoblots (b–f, rabbit; h andcalsequestrin-like proteins (CLP) (b), slow calsequestrin (CSQ) (c),uxiliary a2-subunit of the dihydropyridine receptor (a2-DHPR) (e, io 4 represent microsomal membranes isolated from 21-day-, 44-day-re symbolized by the letters ’d’ and ’y’. Densitometric scanning reve

3% (n 5 3; P , 0.05) between 1.0- and 2.4-year-old muscle specimen4- and 28-month-old rat muscles, respectively. Months are symbolizmarked by arrows. Sizes of molecular mass standards (in kDa) are

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29). Alcoholic myopathy is clearly associated with ab-normal Ca21-ion transients through surface channelsand intracellular ion pumps (30). Muscular central

g rabbit and rat skeletal muscle. Shown are Coomassie-stained gelsrat) labeled with antibodies to fast calsequestrin (CSQ) (b) and

a1-subunit of the dihydropyridine receptor (a1-DHPR) (d, h), thend the a1-subunit of the Na1/K1-ATPase (a1-NKA) (f). (a–f) Lanes 1year-, and 2.4-year-old rabbit muscles, respectively. Days and yearsd a significant decrease of the relative a1-DHPR expression of 24 6g–i) Lanes 1 and 2 represent microsomal membranes isolated fromby the letter ’m’. The position of immunodecorated protein bands isicated on the left.

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core disease was shown to be triggered by mutations inthe ryanodine receptor RyR1 isoform (28, 29). The mus-cle relaxation disorder Brody’s disease is due to pri-mary abnormalities in the sarcoplasmic reticulumCa21-ATPase (28). The major cause for triggering ne-crosis in dystrophic muscle fibres from patients af-flicted with Duchenne muscular dystrophy, limb-girdlemuscular dystrophy or congenital muscular dystrophywas shown to be elevated cytosolic Ca21-levels (31).

ecreased protein–protein interactions and abnormalatios between the Ca21-ATPase and its regulatory

component phospholamban appear to be a major factorin causing decreased Ca21-transients in dilated cardio-

yopathy (32). It is therefore reasonable to assumehat abnormal Ca21-transients may also play a pri-ary or secondary role in sarcopenia, the age-relatedeakness in mammalian skeletal muscle fibres (9).Previous studies on age-related changes in the dihy-

ropyridine receptor focused on the rat animal modelsing binding of pharmacological antagonists to judgeifference in the expression level of this transverseubular protein (33–36). To complement these investi-ations and in order to directly visualize potentiallyltered expression levels and oligomerization, we per-ormed immunobotting and a chemical crosslinkingnalysis of the dihydropyridine receptor. As illustratedn Fig. 1, a relatively comparable protein band patternas observed in microsomal membranes isolated fromifferently aged muscle fibres. Thus to determinehanges in Ca21-handling proteins, their expression

levels had to be determined by the more discriminatingmethod of immunoblotting. Prior to the analysis of thefate of the dihydropyridine receptor, we investigatedpotential shifting in fibre type or isoform expressionpatterns. The fast and slow isoforms of the Ca21-inding protein calsequestrin were employed as mark-rs of fast versus slow muscle characteristics. Whilehe density of fast calsequestrin was only marginallyffected, the relative expression of the slow calseques-rin isoform was clearly up-regulated in older musclebres (Figs. 1b and 1c). Besides the fast calsequestrinonomer of apparent 63 kDa, monoclonal antibodyIIID12 also recognizes high-molecular-mass compo-

nents named calsequestrin-like proteins (Fig. 1b). Thefunction of these proteins is not known. The increase inslow calsequestrin indicates a functional shift to Ca21-handling in the lumen of the sarcoplasmic reticulumwhich is typical for slower-twitching skeletal musclecells.

Most importantly, our immunoblot analysis demon-strated a significantly decreased expression of the a1-subunit of the dihydropyridine receptor in old rabbitand rat muscle as compared to younger specimens(Figs. 1d and 1h). Monoclonal antibody IIID5 immuno-decorated a band of apparent 170 kDa exclusively. Nodegradation products were observed in aging speci-mens. In contrast, the effect of aging was much less

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obvious for the a2-subunit of the dihydropyridine re-ceptor and the a1-subunit of the Na1/K1-ATPase (Figs.1e and 1f).

Since biochemical studies with chemical crosslinkerprobes require larger amounts of muscle tissues foroptimization, we used rabbit muscle homogenates forthe chemical crosslinking study. Increasing amounts ofthe hydrophobic probe DSP of 1.2 nm spacer-armlength caused a general decrease in the monomericreceptor band of approximately 170 kDa (Fig. 2).Higher concentrations of the crosslinker triggered thestabilization of high-molecular-mass complexes above605 kDa. No major differences in the oligomeric statusof the a1-subunit of the dihydropyridine receptor wereobserved between the microsomal fractions isolated

FIG. 2. Chemical crosslinking analysis of the dihydropyridinereceptor in aging rabbit skeletal muscle. Shown are identical immu-noblots labeled with an antibody to the a1-subunit of the dihydro-pyridine receptor (a1-DHPR). Analysis has been performed withmicrosomal membranes isolated from 21-day- (a), 44-day- (b), 1.0-year- (c), and 2.4-year-old (d) rabbit muscles, respectively. Days andyears are symbolized by the letters ’d’ and ’y’. Lanes 1 to 6 represent0, 3, 6, 9, 12, and 25 mg dithiobis-succinimidyl-propionate (DSP) permg protein, respectively. The position of immunodecorated mono-mers is indicated by closed arrows and crosslinking-stabilized high-molecular-mass complexes are marked by open arrows. Sizes ofmolecular mass standards (in kDa) are indicated on the left.

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from differently aged muscles (Fig. 2). The varyingintensity of immunolabeling between the individualsamples is probably due to different degrees ofcrosslinker-induced inhibition of antigen recognition.Since chemical crosslinking introduces covalent bondsbetween receptor subunits, conformational changesmight trigger modifications in epitopes. This in turnmight have an effect on the overall affinity of antibodybinding, especially if monoclonal antibodies are used.

It has been postulated that altered turnover of keyCa21-regulatory membrane proteins of the sarcoplasmicreticulum (37, 38) and uncoupling between the voltagesensor and Ca21-release channel units (33, 34) play animportant role in sarcopenia. The findings presented inthis report agree with the concept that impairment ofexcitation-contraction coupling is one of the underlyingcauses of muscular weakness in the senescent musclefibre (33–36). It appears that a decrease in voltage-sensing dihydropyridine receptor subunits and not ab-normal oligomerization of the transverse-tubular recep-tor and/or impaired triadic receptor coupling triggers areduction in dihydropyridine-sensitive calcium currents(35). Abnormal voltage-sensing finally leads to an overallreduction of the amount of Ca21-ions available for trig-ering mechanical responses in aged skeletal muscle andhus reduces the Ca21-peak transient (33–37). Thus, be-

sides abnormalities in Ca21-uptake mechanisms (39–41),decreased voltage-sensing also appears to contribute tosarcopenia.

ACKNOWLEDGMENTS

Research was supported by a 5th Framework Programme ’Qualityof Life’ project grant from the European Community (QLRT-1999-02034; Investigation on the mechanisms for maintenance and regen-eration in the ageing muscle). The authors thank Kevin P. Campbellfor providing us with monoclonal antibodies to the dihydropyridinereceptor.

REFERENCES

1. Kirkwood, T. B. L., and Austadt, S. N. (2000) Why do we age?Nature 408, 233–238.

2. Finkel, T., and Holbrock, N. J. (2000) Oxidants, oxidative stressand the biology of ageing. Nature 408, 239–247.

3. Brown, M., and Hasser, E. M. (1996) Complexity of age-relatedchange in skeletal muscle. J. Gerontol. 51A, 117–123.

4. Porter, M. M., Vandervoort, A. A., and Lexell, J. (1995) Ageing ofhuman muscle: Structure, function and adaptability. Scand.J. Med. Sci. Sports 5, 129–142.

5. Tseng, B. S., Marsh, D. R., Hamilton, M. T., and Booth, F. W.(1995) Strength and aerobic training attenuate muscle wastingand improve resistance to the development of disability withageing. J. Gerontol. 50A, 113–119.

6. Larsson, L. (1998) The age-related motor disability: Underlyingmechanisms in skeletal muscle at the motor unit, cellular andmolecular level. Acta Physiol. Scand. 163, S27–S29.

7. Lopez-Cepero, J. M., and Sanchez del Pino, M. J. (2001) Skeletalmuscle ageing. Front. Biosci. 6, 26–44.

8. Carlson, B. M. (1995) Factors influencing the repair and adap-

228

vation. J. Gerontol. 50A, 96–100.9. Margareth, A., Damiani, E., and Bortoloso, E. (1999) Sarcoplas-

mic reticulum in aged skeletal muscle. Acta Physiol. Scand. 167,331–338.

0. Squier, T. S., and Bigelow, D. J. (2000) Protein oxidation andage-dependent alterations in calcium homeostasis. Front. Biosci.5, 504–526.

1. Flucher, B. E., and Franzini-Armstrong, C. (1996) Formation ofjunctions involved in excitation-contraction coupling in skeletaland cardiac muscle. Proc. Natl. Acad. Sci. USA 93, 8101–8106.

2. Leong, P., and MacLennan, D. H. (1998) Complex interactionsbetween skeletal muscle ryanodine receptor and dihydropyridinereceptor proteins. Biochem. Cell Biol. 76, 81–694.

3. Froemming, G. R., and Ohlendieck, K. (1998) Oligomerisation ofCa21-regulatory membrane components involved in theexcitation-contraction-relaxation cycle during postnatal develop-ment of rabbit skeletal muscle. Biochim. Biophys. Acta 1387,226–238.

4. Cannell, M. B., and Soeller, C. (1998) Sparks of interest incardiac excitation-contraction coupling. Trends Pharmacol. Sci.19, 16–20.

5. Catterall, W. A. (1995) Structure and function of voltage-gatedion channels. Annu. Rev. Biochem. 64, 493–532.

6. Gurnett, C. A., and Campbell, K. P. (1996) Transmembraneauxiliary subunits of voltage-dependent ion channels. J. Biol.Chem. 271, 27975–27978.

7. Ohlendieck, K. (2000) Changes in Ca21-regulatory membraneproteins during the stimulation-induced fast-to-slow transitionprocess. Bas. Appl. Myol. 10, 99–106.

18. Froemming, G. R., Murray, B. E., Harmon, S., Pette, D., andOhlendieck, K. (2000) Comparative analysis of the isoform ex-pression pattern of Ca21-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequencystimulated muscle fibres. Biochim. Biophys. Acta 1466, 151–168.

9. Pette, D., and Vrbova, G. (1999) What does chronic electricalstimulation teach us about muscle plasticity? Muscle Nerve 22,666–677.

0. Murray, B., and Ohlendieck, K. (1997) Crosslinking analysis ofthe ryanodine receptor and a1dihydropyridine receptor in rabbitskeletal muscle triads. Biochem. J. 324, 689–696.

21. Bradford, M. M. (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72, 248–254.

22. Laemmli, U. K. (1970) Cleavage of bacteriophage T7 early RNAsand proteins on slab gels. Nature 227, 680–685.

23. Dunn, M. J., and Bradd, S. J. (1993) Separation and analysis ofmembrane proteins by SDS-polyacrylamide gel electrophoresis.Meth. Mol. Biol. 19, 203–210.

24. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: Procedure and some applications. Proc. Natl. Acad. Sci.USA 76, 4350–4354.

25. Bradd, S. J., and Dunn, M. J.(1993) Analysis of membrane pro-teins by Western blotting and enhanced chemiluminescence.Methods Mol. Biol. 19, 211–218.

26. Froemming, G. R., and Ohlendieck, K. (2001) Role of ion-regulatory membrane proteins in inherited muscle diseases.Front. Biosci. 6, 65–74.

27. Berchtold, M. W., Brinkmeier, H., and Muntener M. (2000) Cal-cium ion in skeletal muscle: Its crucial role for muscle function,plasticity, and disease. Physiol. Rev. 80, 1215–1265.

28. MacLennan, D. H. (2000) Ca21 signaling and muscle disease.Eur. J. Biochem. 267, 5291–5297.

29. McCarthy, T. V., Quane, K. A., and Lynch, P. J. (2000) Ryano-

3

3

3

3

3

pression of IGF-1 exclusively in skeletal muscle prevents age-


dine receptor mutations in malignant hyperthermia and centralcore disease. Hum. Mutat. 15, 410–417.

30. Preedy, V. R., Patel, V. B., Reilly, M. E., Richardson, P. J.,Falkous, G., and Mantle D. (1999) Oxidants, antioxidants andalcohol: Implications for skeletal and cardiac muscle. Front. Bio-sci. 1, 58–66.

1. Ruegg, U. T., and Gillis, J. M. (2000) Calcium homeostasis indystrophic muscle. Trends Pharmacol. Sci. 20, 351–352.

32. Lennon, N., and Ohlendieck, K. (2001) Impaired Ca21-sequestration in dilated cardiomyopathy (review). Int. J. Mol.Med. 7, 131–141.

3. Delbono, O., O’Rourke, K. S., and Ettinger, W. H. (1995)Excitation-calcium release uncoupling in aged single humanskeletal muscle fibers. J. Membr. Biol. 148, 211–222.

4. Renganathan, M., Messi, M. L., and Delbono, O. (1997) Dihydro-pyridine receptor-ryanodine receptor uncoupling in aged skeletalmuscle. J. Membr. Biol. 157, 247–253.

5. Mayhew, M., Renganathan, M., and Delbono, O. (1998) Effec-tiveness of caloric restriction in preventing age-related changesin rat skeletal muscle. Biochem. Biophys. Res. Commun. 251,95–99.

6. Renganathan, M., Messi, M. L., and Delbono, O. (1998) Overex-

229

related decline in the number of dihydropyridine receptors.J. Biol. Chem. 273, 28845–28851.

37. Damiani, E., Larsson, L., and Margreth, A. (1996) Age-relatedabnormalities in regulation of the ryanodine receptor in ratfast-twitch muscle. Cell Calcium 19, 15–27.

38. Ferrington, D. A., Krainev, A. G., and Bigelow, D. J. (1998)Altered turnover of calcium regulatory proteins of the sarcoplas-mic reticulum in aged skeletal muscle. J. Biol. Chem. 273, 5885–5891.

39. Schoneich, C., Viner, R. I., Ferrington, D. A., and Bigelow, D. J.(1999) Age-related chemical modification of the skeletal musclesarcoplasmic reticulum Ca-ATPase of the rat. Mech. Age. Dev.107, 221–231.

40. Chen, B., Jones, T. E., and Bigelow, D. J. (1999) The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is confor-mationally altered in aged skeletal muscle. Biochemistry 38,14887–14896.

41. Hunter, S. K., Thompson, M. W., Ruell, P. A., Harmer, A. R.,Thom, J. M., Gwinn, T. H., and Adams, R. D. (1999) Humanskeletal sarcoplasmic reticulum Ca21 uptake and muscle func-tion with aging and strength training. J. Appl. Physiol 86, 1858–1865.

Oligomeric Status of the Dihydropyridine Receptor in Aged Skeletal Muscle

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