Peptide Probe Study of the Role of Interaction between the Cytoplasmic and Transmembrane Domains of the Ryanodine Receptor in the Channel Regulation Mechanism
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- 作者：Tomoyo Hamada ; Mark L. Bannister ; Noriaki Ikemoto
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：April 10, 2007
- 年：2007
- 卷：46
- 期：14
- 页码：4272 - 4279
- 全文大小：277K
- 年卷期：v.46,no.14(April 10, 2007)
- ISSN：1520-4995
文摘
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 Ca2+ concentrations (
0.3 
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 Ca2+-dependent channel regulation.In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channelactivation similar to that of DP15.
0.3 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 Ca2+-dependent channel regulation.In further support of this concept, anti-DP15 antibody and cardiac counterpart of DP15 produced channelactivation similar to that of DP15.