Inositol Trisphosphate Receptor and Ca2+ Signalling

Inositol 1,4,5-trisphosphate (InsP₃) is a second messenger that releases Ca²⁺ from the intracellular stores. The InsP₃ receptor (InsP₃-R) was purified and its cDNA was cloned. We have found that InsP₃-R is identical to the P₄₀₀ protein identified as a protein enriched in the cerebellar Purkinje cells. We generated an L fibroblast cell transfectant that produced cDNA derived InsP₃-R. The expressed protein displays high affinity and specificity for InsP₃. InsP₃ induces Ca²⁺ release from the membrane vesicles of the transfected cells. Incorporation of purified InsP₃-R into a lipid bilayer showed InsP₃ induced Ca²⁺ release. These result suggest that InsP₃-R is a Ca²⁺ 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 InsP₃-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 InsP₃ binding. We found novel subtypes of the InsP₃-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 Ca²⁺ release mechanisms, InsP₃-induced Ca²⁺ release (IICR) and Ca²⁺-induced Ca²⁺ release (CICR). Eggs are good materials to analyse the machanism of Ca²⁺ signalling: fertilized hamster eggs exhibit repetitive Ca²⁺ transients as well as the Ca²⁺ wave. A monoclonal antibody to the InsP₃-R inhibited both IICR and CICR respectively upon injection of InsP₃ and Ca²⁺ into hamster eggs. The antibody completely blocked sperm-induced Ca²⁺ waves and Ca²⁺ oscillations. The results indicate that Ca²⁺ release in fertilized hamster eggs is mediated solely by the InsP₃-R, and that Ca²⁺-sensitized IICR, but not CICR, generates Ca²⁺ waves and Ca²⁺ oscillations.