| Budget Amount *help |
¥12,900,000 (Direct Cost: ¥12,900,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥11,500,000 (Direct Cost: ¥11,500,000)
|
|---|
| Research Abstract |
Ca^<2+> release from the sarco/endoplasmic reticulum (S/ER) is an essential process in the intracellular signal transduction mechanism by Ca^<2+>. We estimated the size of lumenal Ca^<2+> -buffering sites in S/ER of bullfrog skeletal muscle and discussed the effect of lumenal Ca^<2+> on the kinetics of Ca^<2+> release. To confirm conclusions, we examined homogeneity of the Ca^<2+> release channels. We already reported that two isoforms of the ryanodine receptor (RyR), α-and β-RyR, coexist in almost equal amounts in bullfrog skeletal muscles and that α-and β-RyR are homologous to types 1 and 3 of mammalian RyR isoforms, respectively. Purified α- and β-RyR were so similar in Ca^<2+> -induced Ca^<2+> release (CICR) activity that they Cannot be differentiated. Their native properties of Ca^<2+> release channels in S/ER, however, remain to be elucidated. With aids of the peculiar property of [^3H]ryanodine binding and immunoprecipitation with a monoclonal antibody to α-RyR, we found that CI
… More
CR activity of α-RyR was specifically suppressed in the native membrane to as low as a few percent of that of the β-RyR.The CICR activity in the skeletal muscle fiber may be primarily ascribed to β-RyR.The S/ER can be regarded a single homogenous compartment in skeletal muscles, whereas mitsugumin29-deficient muscle may have multiple heterogeneous compartments. The complicated time course of IP3-induced Ca^<2+> release from the Ca^<2+> store in smooth muscle and non-muscle cells has controversially been discussed. Those discussions, however, missed the idea which we proposed that the contribution of the efficient free Ca^<2+> concentration gradient across the membrane which was the primary driving force for Ca^<2+> release was larger in higher Ca^<2+> -loaded S/ER than in partially depleted S/ER because of lumenal Ca^<2+> -buffering sites. We are looking for a suitable indicator which reliably reports lumenal Ca^<2+> to further support this idea.
Depletion of Ca_<2+> in the S/ER increases the Ca_<2+> entry through the plasma membrane of smooth muscle or non-muscle cells (store-operated Ca_<2+> entry). we examined whether this store-operated Ca_<2+> entry could be observed in skeletal muscles where the contribution of the IP3 receptor for the Ca_<2+> homeostasis would be minor. The increase in Ca_<2+> entry to refill the Ca_<2+> store and stimulated Mn_<2+> influx through the sarcolemma were successfully observed in intact mouse skeletal muscles. The inflow was similar to reported CRAC current in many respects. Less
|
