Role of the Met3534-Ala4271 region of the ryanodine receptor in the regulation of Ca2+ release induced by calmodulin binding domain peptide.
CaMBP, a peptide corresponding to the 3614-3643 calmodulin (CaM) binding region of the ryanodine receptor (RyR1), is known to activate RyR1 Ca2+ channel. To analyze the mechanism of channel regulation by the CaMBP-RyR1 interaction, we investigated a), CaMBP binding to RyR1, b), induced local conformational changes in the CaMBP binding region of RyR1 using the fluorescent conformational probe badan attached to CaMBP (CaMBP-badan), and c), effects of "a" and "b" on SR Ca2+ release. We also monitored the interaction of CaMBP-badan with CaM and a peptide corresponding to the Met3534-Ala4271 region of RyR1 (R3534-4271) as a control. At lower peptide concentrations (< or =15 microM), CaMBP binding to RyR1 increased the intensity of badan fluorescence emission at a shorter wavelength (the state resembling CaMBP-badan/Ca-CaM) and induced Ca2+ release. Further increase in CaMBP concentration (up to approximately 50 microM) produced more binding of CaMBP accompanied by further increase in the badan fluorescence emission but at a longer wavelength (the state resembling CaMBP-badan/apo-CaM) and inhibited Ca2+ release. Binding of CaMBP-badan to R3534-4271 increased the intensity of badan fluorescence, showing the similar concentration-dependent red-shift of the emission maximum. It is proposed that CaMBP interacts with two classes of binding sites located in the Met3534-Ala4271 region of RyR1, which activate and inhibit the Ca2+ channel, respectively.
Gangopadhyay
Jaya Pal
JP
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Ikemoto
Noriaki
N
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium