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Papers In Press, published online ahead of print October 6, 2004
Department of Anesthesia Research, Boston Biomedical Research Institute, Watertown, MA 02472
Corresponding Author: fessenden{at}bbri.org
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 4081-4092 (EF1) and 4116-4127 (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 to wtRyR1. However, in [3H]ryanodine binding measurements, both Ca2+ activation and inhibition of the EF1 mutant was significantly altered compared to 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 4254-4265 (EF3), 4407-4418 (EF4) or 4490-4502 (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 to wtRyR1. However, in [3H]ryanodine binding experiments, the Ca2+-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected compared to wtRyR1.
J. Biol. Chem, 10.1074/jbc.M411136200
Submitted on September 28, 2004
Revised on October 6, 2004
Accepted on October 6, 2004
Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1
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