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J Biol Chem, Vol. 274, Issue 37, 25971-25974, September 10, 1999

COMMUNICATION
Molecular Identification of the Ryanodine Receptor Pore-forming Segment

Mingcai Zhao, Pin Li, Xiaoli Li, Lin Zhang, Robert J. Winkfein^¶, and S. R. Wayne Chen

From the  Cardiovascular Research Group, Department of Physiology and Biophysics, and Department of Biochemistry and Molecular Biology, and the ^¶ Medical Research Council Group on Ion Channels and Transporters, University of Calgary, Calgary, Alberta T2N 4N1, Canada

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca²⁺ release measurements, [³H]ryanodine binding, and single channel recordings in planar lipid bilayers. We report here that single point mutations introduced into this region of the mouse cardiac ryanodine receptor reduce or abolish high affinity [³H]ryanodine binding. Single channel analysis revealed that a single substitution of alanine for glycine 4824 within this region reduced the single channel conductance by 97%, from 798 picosiemens (pS) for the wild type channel to 22 pS. The G4824A mutant channel was modulated by Ca²⁺, Mg²⁺, ATP, caffeine, ruthenium red, and ryanodine. Co-expression of the wild type and G4824A mutant proteins produced single channels that have intermediate unitary conductances of 516, 256, 176, and 60 pS. These data suggest that this conserved region constitutes an essential part of the ryanodine binding site and the channel conduction pathway of the ryanodine receptor.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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