Conformation-dependent stability of junctophilin 1 (JP1) and Ryanodine Receptor Type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols

Andrew J. Phimister, Jozsef Lango, Hui Lee Eun, Michael A. Ernst-Russell, Hiroshi Takeshima, Ma Jianjie, Paul D. Allen, Isaac N Pessah

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4′- maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 μM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H 2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin- thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.

Original languageEnglish (US)
Pages (from-to)8667-8677
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number12
DOIs
StatePublished - Mar 23 2007

ASJC Scopus subject areas

  • Biochemistry

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