INOSITOL TRISPHOSPHATE RECEPTOR AND CA2+ SIGNALING

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.