Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry

Andrew A. Voss, Jozsef Lango, Michael Ernst-Russell, Dexter Morin, Isaac N Pessah

Research output: Contribution to journalArticle

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Abstract

The skeletal-type ryanodine receptor (RyR1) undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and non-covalent interactions driven by the transmembrane redox potential that enable redox sensing. Tight redox regulation of RyR1 is thought to be primarily mediated through highly reactive (hyperreactive) cysteines. Of the 100 cysteines per subunit of RyR1, ∼25-50 are reduced, with 6-8 considered hyperreactive. Thus far, only Cys-3635, which undergoes selective adduction by NO, has been identified. In this report, RyR1-enriched junctional sarcoplasmic reticulum is labeled with 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM, 1 pmol/μg of protein) in the presence of 10 mM Mg2+, conditions previously shown to selectively label hyperreactive sulfhydryls and eliminate redox sensing. The CPM-adducted RyR1 is separated by gel electrophoresis and subjected to in-gel tryptic digestion. Isolation of CPM-adducted peptides is achieved by analytical and microbore high-performance liquid chromatography utilizing fluorescence and UV detection. Subsequent analysis using two direct and one tandem mass spectrometry methods results in peptide masses and sequence data that, compared with the known primary sequence of RyR1, enable unequivocal identification of CPM-adducted cysteines. This work is the first to directly identify seven hyperreactive cysteines: 1040, 1303, 2436, 2565, 2606, 2611, and 3635 of RyR1. In addition to Cys-3635, the nitrosylation site, six additional cysteines may contribute toward redox regulation of the RyR1 complex.

Original languageEnglish (US)
Pages (from-to)34514-34520
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number33
DOIs
StatePublished - Aug 13 2004

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Ryanodine Receptor Calcium Release Channel
Mass spectrometry
Cysteine
Mass Spectrometry
Oxidation-Reduction
Nitric Oxide
Gels
Peptides
High performance liquid chromatography
Sarcoplasmic Reticulum
Tandem Mass Spectrometry
Electrophoresis
Membrane Potentials
Cations
Labels
Digestion
Fluorescence
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry. / Voss, Andrew A.; Lango, Jozsef; Ernst-Russell, Michael; Morin, Dexter; Pessah, Isaac N.

In: Journal of Biological Chemistry, Vol. 279, No. 33, 13.08.2004, p. 34514-34520.

Research output: Contribution to journalArticle

Voss, Andrew A. ; Lango, Jozsef ; Ernst-Russell, Michael ; Morin, Dexter ; Pessah, Isaac N. / Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 33. pp. 34514-34520.
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