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J. Biol. Chem., Vol. 279, Issue 51, 53028-53035, December 17, 2004

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Mutational Analysis of Putative Calcium Binding Motifs within the Skeletal Ryanodine Receptor Isoform, RyR1^*

James D. Fessenden, Wei Feng¶, Isaac N. Pessah¶, and P. D. Allen

From the Department of Anesthesia Research, Brigham and Women's Hospital, Boston, Massachusetts 02115 and the ^¶Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, California 95616

The functional relevance of putative Ca²⁺ binding motifs previously identified with Ca²⁺ overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca²⁺-imaging techniques, [³H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [³H]ryanodine binding measurements, both Ca²⁺ activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [³H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca²⁺ concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3–5) did not affect functional responses in 1B5 myotubes except that the EC₅₀ for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [³H]ryanodine binding experiments, the Ca²⁺-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

Received for publication, September 28, 2004

^* 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.

To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7886; Fax: 617-972-1761; E-mail: fessenden{at}bbri.org.

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