|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
Excitation-contraction coupling in mammalian skeletal muscles is generally understood as follows : dihydropyridine receptor (DHPR), the voltage sensor to detect the change in the membrane potential in the sarcolemma, transmits some signals to the ryanodine receptor (RyR), primarily RyR1 isoform, on the junctional sarcoplasmic reticulum, resulting in the Ca^<2+> release through RyR form the Ca^<2+> store. In frog skeletal muscles, however, two isoforms of RyR,alpha-and beta-RyR,were found in nearly equal amount. Our present project is to know whether the voltage sensor(s) should be single corresponding to alpha-RyR or dual to alpha- and beta-RyR.
Initially we confirmed that the microsomal fraction showed [^3H]PN200-100 binding activity, indicating that DHPR is likely also to be the voltage sensor in frog skeletal muscle as is true of mammals. The we concentrated on the cloning of alpha_<IS> subunit of frog DHPR.cDNA library (2*10^<10> pfu/ml) was prepared using oligo (dT) primer from mRNA isolated from frog leg muscles. A synthesized oligomeric primer, of which the seuence was determined from aligned DNA Sequences of rabbit, mouse and carp alpha_<IS> subunits, allowed us to isolate a 700 bp fragment from total RNA of frog skeletal muscle. The sequence was highly homologous to those of the alpha_<IS> subunits which were already reported. Northern blot analysis with the probe of this 700 bp fragment showed a single band of about 6kb. Following screening of the cDNA library with this probe and mapping with EcoRI,24 independent clones were obtained. Determination of the sequences of these clones is now in progress. We will further proceed to clone and sequence beta_1 and the other kinds of subunits.