Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin.
The fluorogenic sulfhydryl probe 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) (1-50 nM) is used to characterize the functional role and location of highly reactive thiol groups on the ryanodine-sensitive Ca2+ release channel complex [i.e., ryanodine receptors (RyRs)] of skeletal and cardiac junctional sarcoplasmic reticulum (SR). The kinetics of forming fluorescent CPM adducts with junctional but not longitudinal SR membrane proteins (0.02-1 pmol of CPM/microgram of SR protein) are found to be markedly dependent on the presence of physiological and pharmacological modulators of the RyR Ca2+ channel. RyR agonists, micromolar Ca2+, and nanomolar ryanodine promote a slow SR thiol-CPM reaction, with an apparent rate constant k of 0.0021 +/- 0.0002 sec-1, and > 89% of the fluorescence is associated with the 110-kDa Ca2+ pump, which constitutes 68% of the protein in the SR preparations. However, in the presence of Ca2+ channel antagonists (millimolar Mg2+, millimolar Ca2+, or micromolar ryanodine), CPM rapidly forms adducts with a single class of highly reactive (hyperreactive) SR thiols (k = 0.025 +/- 0.002 sec-1). Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CPM-labeled SR protein and Western blot analyses with antiryanodine or antitriadin antibodies reveal that the hyperreactive thiols labeled by CPM under conditions favoring channel closure are localized principally to the RyR protomer and triadin, which constitute < 6% of the protein in the SR preparation. Immunoprecipitation experiments with antiryanodine and antitriadin monoclonal antibodies confirm the location of CPM-labeled thiol groups on RyR and triadin, respectively. The results indicate that the RyR and triadin contain a small number of highly reactive cysteine residues that selectively conjugate with CPM only when channel closure is favored. It is shown that either 1) the redox state (sulfhydryl/disulfide status) or 2) the accessibility of the hyperreactive thiols on the RyR and triadin is determined by the conformational state of the channel. Covalent modification of hyperreactive thiols with nanomolar CPM inhibits both Ca(2+)-induced Ca2+ release and the gating activity of single channels reconstituted in bilayers, revealing the essential functional importance of hyperreactive thiols on channel-associated proteins. 1,4-Naphthoquinone (0.4-40 pmol/micrograms of protein) selectively oxidizes hyperreactive thiols on RyR and triadin and releases Ca2+ from SR vesicles, without inhibiting Ca(2+)-ATPase activity. The results provide direct evidence of the existence and functional role of hyperreactive cysteine residues on the RyR and triadin in regulating the gating of ryanodine-sensitive intracellular Ca2+ channels and strongly suggest that these important Ca2+ regulatory channels may be an important target for oxidative cell damage mediated by quinones.
Liu
G
G
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis 95616.
Abramson
J J
JJ
Zable
A C
AC
Pessah
I N
IN
D000818:Animals; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002352:Carrier Proteins; D003374:Coumarins / Q000737:chemistry*; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009124:Muscle Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000502:physiology*
D015220:Calcium Channels; D002352:Carrier Proteins; D003374:Coumarins; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013438:Sulfhydryl Compounds; C498271:Trdn protein, rat; C070048:triadin; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; D017301:Ca(2+) Mg(2+)-ATPase