Structure and function of ryanodine receptors

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The structure-function relationship of ryanodine receptor subtypes (namely, RyR-l, 2 and 3) have been investigated by means of molecular biological and genetic methords, for example in vitro mutagenesis and generation of knockout mice. Our major findings in this research project are listed below.1. Generation and characterization of mutant mice lacking RyR-2. Because function of RyR-2 was unknown in embryonic heart, we have generated mutant mice lacking RyR-2. The results in the mutant mice showed non-essential function of RyR-2 for contraction of embryonic cardiac muscle cells, however, the RyR-2-deficient mice exhibiting cardiac arrest die around embryonic day 10. Our results obtained indicate that RyR-2 is essential for the maintenance of intracellular Ca^<2+> stores by regulating luminal resting Ca^<2+> levels in embryonic cardiac muscle cells.2. Intramolecular functional regions on the primary structure of the ryanodine receptor. We have mapped a region critical for skeletal muscle excitation-contraction coupling to the called D2 region on the RyR-1 molecule in our cDNA expression system using RyRs-deficient muscle. By combination of cDNA expression and single channel measurement, we have located the Ca^<2+> release channel-forming domain and a portion (designated as D3 region) responsible for Ca^<2+> binding for channel inactivation on the RyR-1 molecule.3. Identification of novel proteins localized in skeletal muscle triad junction. We have identified mitsugumin29 (MG29) and MG23 as novel transmembrane proteins from skeletal muscle the triad junction by isolating specific monoclonal antibodies and cloning cDNAs. MG29 was shown to be a novel member of the synaptophysin family, suggesting that the presynaptic region and triad junction share evolutionally related components.