title: Interaction of the Lys(3614)-Asn(3643) calmodulin-binding domain with the Cys(4114)-Asn(4142) region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation.
abstract: In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.
authors.affiliation: Boston Biomedical Research Institute, Watertown, MA 02472, USA.
authors.forename: Jaya P
authors.initials: JP
authors.lastname: Gangopadhyay
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ikemoto
mesh_terms: D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000819:agonists* / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D001216:Asparagine; D008239:Lysine; D003545:Cysteine; D002118:Calcium