INVOLVEMENT OF THE C-TERMINUS OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN CA-2+ RELEASE ANALYZED USING REGION-SPECIFIC MONOCLONAL-ANTIBODIES

We have studied the effects of monoclonal antibodies that recognize different epitopes of the cerebellar Ins(1,4,5)P3 receptor on Ins(1,4,5)P3-induced Ca2+-release activity. Ins(1,4,5)P3 stimulated Ca2+ flux from cerebellar microsomes, and half-maximal Ca2+ release occurred at 112 +/- 8 nM-Ins(1,4,5)P3 [concentration causing half-maximal effect (EC50) = 112.8 nM]. The minimum concentration of Ins(1,4,5)P3 necessary to initiate Ca2+ release (threshold concentration) was 20 +/- 5 nM. A monoclonal antibody (mAb) 18A10 (50-mu-g/ml), which recognizes the C-terminal region of the Ins(1,4,5)P3 receptor, suppressed Ins(1,4,5)P3-induced Ca2+ release: the EC50 and threshold concentration shifted to 460 +/- 56 nM and 61 +/- 6 nM respectively. On the other hand, the antibody at the same concentration raised the affinity of the receptor for binding to Ins(1,4,5)P3, and the K(d) value decreased from 43 +/- 12 nM to 25 +/- 4 nM without a change in the number of Ins(1,4,5)P3-binding sites. However, mAbs that recognize the N-terminal domain affected neither Ca2+ release nor Ins(1,4,5)P3 binding. Among the various synthetic peptides, only the 12-residue-long peptide from the most C-terminal portion of the receptor (amino acid residues 2736-2747) reacted strongly with mAb18A10. From these findings, combined with the Immunogold localization of the cerebellar Ins(1,4,5)P3 receptor [Otsu, Yamamoto, Maeda, Mikoshiba & Tashiro (1990) Cell Struct. Funct. 15, 163-173]; we concluded that the C-terminus of the Ins(1,4,5)P3 receptor is exposed to the cytoplasmic side of the smooth endoplasmic reticulum and plays an important role in the regulation of both Ins(1,4,5)P3-binding affinity and channel gating.