Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseasesare localized to three easily definable regions: region 1 (N-terminal domain), region 2 (central domain),and a rather broad region 3 containing the channel pore. As shown in our recent studies, the interdomaininteraction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here wepresent evidence that within region 3 there is a similar channel regulation mechanism mediated by aninterdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significantactivation of [
3H]ryanodine binding above threshold Ca
2+ concentrations (
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0.3
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M), but MH mutations(L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identifythe DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and aphotoaffinity cross-linker APG) was cross-linked to RyR1, and the site of cross-linking was identified bygel analysis of fluorescently labeled proteolytic fragments with the aid of Western blotting with site-specific antibodies. The shortest fluorescently labeled band was a 96 kDa fragment which was stainedwith an antibody directed to the region of residues 4114-4142 of RyR1, indicating that the interactionbetween the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containingthe region of residues 4114-4142 is involved in the mechanism of Ca
2+-dependent channel regulation.In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channelactivation similar to that of DP15.