|Budget Amount *help
¥9,500,000 (Direct Cost : ¥9,500,000)
Fiscal Year 1997 : ¥4,500,000 (Direct Cost : ¥4,500,000)
Fiscal Year 1996 : ¥5,000,000 (Direct Cost : ¥5,000,000)
1. We found that the "core" region for IP3 binding in the type 1 IP3 receptor (IP3R) is located in the N-terminal region from 225 to 578 a.a., and that within this region, at least three basic amino acids (Arg-265, Lys-508, Arg-511) are essential for binding to the negatively-charged IP3 molecule.
2. The IP3 binding activity of type 1 and 3 expressed in Sf9 cells displayd an opposite dependency to increasing Ca2+ ; affinity for IP3 is decreased in type 1 (EC50=100nM Ca2+), whereas increased in type 3 (872nM). IP3 sensitivity in IP3-induced Ca2+ release (IICR) of type 3 was lower than that of type 1 (EC50=358nM vs 226nM IP3). These suggest that cytosolic Ca2+ as well aw IP3 concentrations are involved in type-specific IICR.
3. Type 2 has been thought to be "high affinity" IP3R.However, liver IP3R of type 1 knock-out mice, in which type 2 predominates, showed a similar IP3 sensitivity in IICR as type 1.
4. By expressing a fusion protein with green fluorescent protein (GFP) in Xenopus oocyte
s, we showed that the expressed C-terminal region including a putative channel region is sufficient for the ER localization and the tetramer formation.
5. We established transgenic mouse lines carrying beta-gal gene controlled by type 1 gene promoter, and analyzed the gene expression by detecting beta-gal activity in the nervous system. We identified two cerebellum-specific regions in this promoter ; one is a positive element from -398--295, the other is an E-box-like box I from -334--318. We also determined the primary structure of type 2 gene promoter, and found the presence of multiple transcription initiation sites.
6. By immunohistochemistry of rat kidney, we localized type 1 IP3R to vascular smooth muscle cells and mesangial cells, type 2 to intercalated cells of the collecting duct, and type 3 to vascular smooth muscle cells, mesangial cells and principal cells of the cortical collecting duct. Intracellularly, type 1 and 2 are located throughout the cytoplasm, whereas type 3 is restricted to the basolateral side, suggesting the differential Ca2+ signaling due to the polarization in the subcellular localization of distinct types. Less