Molecular Basis of Ca2+ Binding to the Ryanodine Receptor for Channel Activation

[Speaker] Takashi Murayama:1
[Co-author] Haruo Ogawa:2, Nagomi Kurebayashi:1, Seiko Ohno:3, Minoru Horie:3, Takashi Sakurai:1
1:Department of Pharmacology, Juntendo University School of Medicine, Japan, 2:Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan, 3:Department of Cardiovascular Medicine, Shiga University of Medical Science, Japan

Ryanodine receptors (RyRs) are Ca2+ release channels in the sarcoplasmic reticulum of skeletal and cardiac muscles and are essential for muscle contraction. It is well known that RyR channels are activated by Ca2+ and that caffeine, a xanthine derivative, enhances the Ca2+ sensitivity. However, it remains largely unknown about the actual molecular mechanisms by these ligands. Here, we report the molecular basis of Ca2+ binding to RyRs, the initial step for channel activation, and its regulation by caffeine-binding site. RyR1 and RyR2 carrying mutations in the putative Ca2+- and caffeine-binding sites were functionally analyzed and the results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We verified the proposed binding sites to be the actual binding sites. Surprisingly, a single hydrophobic interaction between tryptophan and isoleucine in the caffeine-binding site normally reduces the Ca2+ sensitivity of the Ca2+-binding site by increasing the size of Ca2+-binding pocket, and caffeine enhances the Ca2+ sensitivity by breaking the interaction to make the Ca2+-binding pocket smaller which is more suitable for Ca2+ binding. Mutations associated with arrhythmogenic heart diseases in RyR2 at the tryptophan and nearby residues also break the interaction to enhance the Ca2+ sensitivity. These results suggest that the caffeine-binding site is an important physiological regulator for the Ca2+ sensitivity of the RyR channels and also implicated in disease states.
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