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Originally published In Press as doi:10.1074/jbc.M609936200 on January 19, 2007

J. Biol. Chem., Vol. 282, Issue 12, 8667-8677, March 23, 2007
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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{ddagger}1, Jozsef Lango{ddagger}, Eun Hui Lee§, Michael A. Ernst-Russell{ddagger}, Hiroshi Takeshima||, Jianjie Ma**, Paul D. Allen§, and Isaac N. Pessah{ddagger}2

From the {ddagger}Department of Veterinary Molecular Biosciences and Center for Children's Environmental Health and Disease Prevention, University of California, Davis, California 95616, the §Department of Anesthesia Research, Brigham & Women's Hospital, Boston, Massachusetts 02115, the Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea, the ||Department of Biochemistry, Tohoku University, Sendai 980-8575, Japan, and the **Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

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, H2O2, 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.


Received for publication, October 23, 2006 , and in revised form, January 3, 2007.

* This work was supported by National Institutes of Health Grants 2RO1-AR43140, 2PO1-AR17605 (to P. D. A. and I. N. P.), and 1PO1-ES11269 (to I. N. P. and J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Present address: Novartis Pharmaceuticals, 4560 Horton St., Emeryville, CA 94608.

2 To whom correspondence should be addressed. Tel.: 530-752-6696; Fax: 530-752-4698; E-mail: inpessah{at}ucdavis.edu.


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