|Budget Amount *help
¥12,900,000 (Direct Cost : ¥12,900,000)
Fiscal Year 1999 : ¥4,900,000 (Direct Cost : ¥4,900,000)
Fiscal Year 1998 : ¥8,000,000 (Direct Cost : ¥8,000,000)
Type 1 inositol 1, 4, 5-trisphosphate receptor (IP, R1), an inositol 1, 4, 5-trisphosphate (IPィイD23ィエD2-gated CaィイD12+ィエD1 release channel binds IPィイD23ィエD2 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IPィイD23ィエD2 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IPィイD23ィエD2, binding constructs expressed in E. coli significantly affect their production as soluble protein Residues 1-6O4 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IPィイD23ィエD2 of T604 (KィイD2dィエD2 = 45nM) is comparable to of the native IPィイD23ィエD2R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IPィイD23ィエD2R1 for IPィイD23ィエD2.
The dependency of purified mouse cerebellar type 1 inositol, 1, 4, 5-trisphosphate receptor (IPィイD23ィエD2R1)/CaィイD22+ィエD2 channel function on cytoplasmic CaィイD12+ィエD1 was examined. In contrast to the channels in crude systems, the purified IPィイD23ィエD2R1 reconstituted into planar lipid bilaryers did not show the bell-shaped dependence on CaィイD12+ィエD1. It was activated with increasing CaィイD12+ィエD1 sublinearly without inhibition even up to 2000 μM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high CaィイD12+ィエD1 concentrations. Calmodulin antagonists reversed the CaィイD12+ィエD1 -dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic CaィイD12+ィエD1 is not an intrinsic property of the IPィイD23ィエD2R1, and the CaィイD12+ィエD1 ?dependent inactivation is directly mediated by calmodulin.