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J. Biol. Chem., Vol. 280, Issue 44, 36994-37004, November 4, 2005

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Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion^*

Alanna M. Hurne1, Jennifer J. O'Brien12, Douglas Wingrove¶, Gennady Cherednichenko, Paul D. Allen¶, Kurt G. Beam, and Isaac N. Pessah3

From the Department of Molecular Biosciences, School of Veterinary Medicine and Center for Children's Environmental Health and Disease Prevention, University of California, Davis, California 95616, the Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, and the ^¶Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca²⁺ entry channel with SOCC-like properties, has recently been presented (Cherednichenko, G., Hurne, A. M., Fessenden, J. D., Lee, E. H., Allen, P. D., Beam, K. G., and Pessah, I. N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15793-15798). This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the _1s-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca²⁺ channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca²⁺>Sr²⁺Ba²⁺, and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys⁴⁹⁵⁸ and Cys⁴⁹⁶¹ within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Received for publication, June 14, 2005 , and in revised form, August 5, 2005.

^* This work was supported by National Institutes of Health Grants 2P0 AR17605, 1P0ES11269, and 2R01 AR43140. 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 Both authors made equal contributions to this work.

² Present address: Dept. of Pharmacology, University of Washington, Seattle, WA 98195.

³ To whom correspondence should be addressed: Dept. of VM: Molecular Biosciences, University of California, Davis, CA 95616. Tel.: 530-752-6696; Fax: 530-752-4698; Email: inpessah{at}ucdavis.edu.

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