Spectroscopic monitoring of local conformational changes during the intramolecular domain-domain interaction of the ryanodine receptor

Biochemistry. 2002 Feb 5;41(5):1492-501. doi: 10.1021/bi015581z.


The amino (N)-terminal and central regions of the ryanodine receptor (RyR) containing most mutation sites of malignant hyperthermia (MH) and central core disease (CCD) seem to be involved in the Ca(2+) channel regulation. Our recent peptide probe study (Yamamoto, T., El-Hayek, R., and Ikemoto, N. (2000) J. Biol. Chem. 275, 11618-11625) suggested the hypothesis that a close contact between the N-terminal and central domains (zipping) stabilizes the closed-state of the channel, while removal of the contact (unzipping) deblocks the channel, causing channel-activation effects. We here report the results of our recent effort to monitor local conformational changes in the putative domain-domain interaction site to test this hypothesis. The conformation-sensitive fluorescence probe, methyl coumarin acetamide (MCA), was incorporated into RyR in a protein- and site-specific manner by using DP4 (the peptide corresponding to the Leu(2442)-Pro(2477) region of the central domain) as a site-directing carrier. The site of MCA labeling was localized in the 150 kDa N-terminal region of RyR, indicating that DP4 and its in vivo counterpart (a portion of the central domain) interact with the N-terminal region. RyR-activating domain peptides, DP4 and DP1 (corresponding to the Leu(590)-Cys(609) region of the N-terminal domain), and depolarization of the T-tubule moiety of the triad (physiologic stimulation) induced a rapid decrease in the fluorescence intensity of the protein-bound MCA and Ca(2+) release at a somewhat slower rate. The accessibility of the protein-bound MCA to the fluorescence quencher was increased in the presence of DP4. These results are all consistent with the above hypothesis.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Azides / metabolism
  • Binding, Competitive / genetics
  • Cross-Linking Reagents / metabolism
  • Fluorescence Polarization / methods
  • Membrane Potentials / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Binding / genetics
  • Protein Conformation
  • Protein Structure, Tertiary / genetics
  • Rabbits
  • Ryanodine Receptor Calcium Release Channel / chemistry*
  • Ryanodine Receptor Calcium Release Channel / genetics
  • Ryanodine Receptor Calcium Release Channel / metabolism*
  • Spectrometry, Fluorescence / methods
  • Succinimides / metabolism


  • Azides
  • Cross-Linking Reagents
  • Peptides
  • Ryanodine Receptor Calcium Release Channel
  • Succinimides
  • sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate