title: Peptide probe study of the role of interaction between the cytoplasmic and transmembrane domains of the ryanodine receptor in the channel regulation mechanism.
abstract: Ryanodine receptor (RyR) mutations linked with some congenital skeletal and cardiac diseases are 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 interdomain interaction between regions 1 and 2 plays a critical role in channel regulation and pathogenesis. Here we present evidence that within region 3 there is a similar channel regulation mechanism mediated by an interdomain interaction. DP15, a peptide corresponding to RyR1 residues 4820-4841, produced significant activation of [3H]ryanodine binding above threshold Ca2+ concentrations (>or=0.3 microM), but MH mutations (L4823P or L4837V) made in DP15 almost completely abolished its channel activating function. To identify the DP15 binding site(s) within RyR1, DP15 (labeled with a fluorescent probe Alexa Fluor 680 and a photoaffinity cross-linker APG) was cross-linked to RyR1, and the site of cross-linking was identified by gel 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 stained with an antibody directed to the region of residues 4114-4142 of RyR1, indicating that the interaction between the region of residues 4820-4841 adjacent to the channel pore and the 96 kDa segment containing the 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 channel activation similar to that of DP15.
authors.affiliation: Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Hamada
authors.forename: Mark L
authors.initials: ML
authors.lastname: Bannister
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ikemoto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D012519:Sarcoplasmic Reticulum
chemical_list: D000906:Antibodies; D015220:Calcium Channels; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium