Conformation-dependent Stability of Junctophilin 1 ( JP1) and Ryanodine Receptor Type 1 (RyR1) Channel Complex Is Mediated by Their Hyper-reactive Thiols.
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 Mg(2+)) compared with conditions that promote high channel activity (e.g. 100 mum Ca(2+), 10 mm caffeine, 5 mm ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H(2)O(2), NO, or O(2), 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.[1]References
- Conformation-dependent Stability of Junctophilin 1 (JP1) and Ryanodine Receptor Type 1 (RyR1) Channel Complex Is Mediated by Their Hyper-reactive Thiols. Phimister, A.J., Lango, J., Lee, E.H., Ernst-Russell, M.A., Takeshima, H., Ma, J., Allen, P.D., Pessah, I.N. J. Biol. Chem. (2007) [Pubmed]
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