Inositol trisphosphate receptor and Ca 2+ signalling

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Abstract
Inositol 1,4,5-trisphosphate (InsP$_{3}$) is a second messenger that releases Ca$^{2+}$ from the intracellular stores. The InsP$_{3}$ receptor (InsP$_{3}$-R) was purified and its cDNA was cloned. We have found that InsP$_{3}$-R is identical to the P$_{400}$ protein identified as a protein enriched in the cerebellar Purkinje cells. We generated an L fibroblast cell transfectant that produced cDNA derived InsP$_{3}$-R. The expressed protein displays high affinity and specificity for InsP$_{3}$. InsP$_{3}$ induces Ca$^{2+}$ release from the membrane vesicles of the transfected cells. Incorporation of purified InsP$_{3}$-R into a lipid bilayer showed InsP$_{3}$ induced Ca$^{2+}$ release. These result suggest that InsP$_{3}$-R is a Ca$^{2+}$ release channel. Immunogold method using monoclonal antibodies against the receptor showed that it is highly condensed on the smooth surfaced endoplasmic reticulum (ER) and slightly on the outer nuclear membrane and rough ER. Cross linking experiments show that the InsP$_{3}$-R forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila melanogaster, and this region has the critical sequences for InsP$_{3}$ binding. We found novel subtypes of the InsP$_{3}$-R resulting from RNA-splicing that are expressed in a tissue-specific and developmentally specific manner and also resulting from different genes. It is believed that there are two Ca$^{2+}$ release mechanisms, InsP$_{3}$-induced Ca$^{2+}$ release (IICR) and Ca$^{2+}$-induced Ca$^{2+}$ release (CICR). Eggs are good materials to analyse the machanism of Ca$^{2+}$ signalling: fertilized hamster eggs exhibit repetitive Ca$^{2+}$ transients as well as the Ca$^{2+}$ wave. A monoclonal antibody to the InsP$_{3}$-R inhibited both IICR and CICR respectively upon injection of InsP$_{3}$ and Ca$^{2+}$ into hamster eggs. The antibody completely blocked sperm-induced Ca$^{2+}$ waves and Ca$^{2+}$ oscillations. The results indicate that Ca$^{2+}$ release in fertilized hamster eggs is mediated solely by the InsP$_{3}$-R, and that Ca$^{2+}$-sensitized IICR, but not CICR, generates Ca$^{2+}$ waves and Ca$^{2+}$ oscillations.