Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls

Guohua Liu, Isaac N Pessah

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Abstract

The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics >10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (≤40 pmol/μg protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by β-mercaptoethanol or dithiothreitol into its component RyR1 and triadin protomers. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1 triadin complex can signal information across the SR membrane.

Original languageEnglish (US)
Pages (from-to)33028-33034
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number52
StatePublished - Dec 30 1994

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Ryanodine Receptor Calcium Release Channel
Molecular interactions
Oxidation-Reduction
Glycoproteins
Sarcoplasmic Reticulum
Molecular Weight
Molecular weight
Ryanodine
Labeling
Anti-Idiotypic Antibodies
triadin
Ruthenium Red
Neomycin
Mercaptoethanol
Antibodies
Dithiothreitol
Protein Subunits
Caffeine
Electrophoresis
Immunoprecipitation

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{24b6d48ef8e640c99be10a7e1846aadc,
title = "Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls",
abstract = "The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics >10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (≤40 pmol/μg protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by β-mercaptoethanol or dithiothreitol into its component RyR1 and triadin protomers. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1 triadin complex can signal information across the SR membrane.",
author = "Guohua Liu and Pessah, {Isaac N}",
year = "1994",
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T1 - Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls

AU - Liu, Guohua

AU - Pessah, Isaac N

PY - 1994/12/30

Y1 - 1994/12/30

N2 - The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics >10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (≤40 pmol/μg protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by β-mercaptoethanol or dithiothreitol into its component RyR1 and triadin protomers. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1 triadin complex can signal information across the SR membrane.

AB - The fluorogenic maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin (CPM) has been shown to selectively form Michael adducts with hyperreactive sulfhydryls on the skeletal sarcoplasmic reticulum (SR) ryanodine receptor (RyR1) and triadin which are essential for normal Ca2+ channel function (Liu, G., Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200). The present report demonstrates a functionally important interaction between RyR1 and triadin which involves, in part, redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation. Nanomolar CPM is shown to selectively label RyR1 and triadin only in the presence of Ca2+ channel inhibitors (Mg2+, neomycin, ruthenium red, or anti-triadin antibody). Treatment of SR with channel activators (micromolar Ca2+, nanomolar ryanodine, or millimolar caffeine), 1) slows CPM labeling kinetics >10-fold, 2) negates CPM labeling of channel-associated sulfhydryls, and 3) stabilizes a high molecular weight complex (HMWC) which appears on nonreducing SDS-polyacrylamide gel electrophoresis gels. The HMWC is positively identified as RyR1 and triadin by Western blot and immunoprecipitation analyses. High-affinity [3H]ryanodine-binding sites are immunoprecipitated by either anti-RyR1 or anti-triadin antibody dose dependently. 1,4-Naphthoquinone (≤40 pmol/μg protein) selectively oxidizes hyperreactive sulfhydryls on RyR1 and triadin, induces Ca2+ efflux from SR, and stabilizes the HMWC. The HMWC is reduced by β-mercaptoethanol or dithiothreitol into its component RyR1 and triadin protomers. The results provide direct evidence for the existence of a functionally important complex between RyR1 and triadin whose stability is determined by the redox state of hyperreactive sulfhydryl moieties which are allosterically regulated by physiological and pharmacological channel ligands. The present results suggest a possible molecular mechanism by which localized transient changes in the redox state within the RyR1 triadin complex can signal information across the SR membrane.

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