INOSITOL TRISPHOSPHATE RECEPTOR AND CA2+ SIGNALING

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releas es Ca2+ from the intracellular stores. The InsP3 receptor (InsP3-R) wa s purified and its cDNA was cloned. We have found that InsP3-R is iden tical to the P400 protein identified as a protein enriched in the cere bellar Purkinje cells. We generated an L fibroblast cell transfectant that produced cDNA derived InsP3-R. The expressed protein displays hig h affinity and specificity for InsP3. InsP3 induces Ca2+ release from the membrane vesicles of the transfected cells. Incorporation of purif ied InsP3-R into a lipid bilayer showed InsP3 induced Ca2+ release. Th ese 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 linkin g experiments show that the InsP3-R forms a homotetramer. The approxim ately 650 N-terminal amino acids are highly conserved between mouse an d Drosophila melanogaster, and this region has the critical sequences for InsP3 binding. We found novel subtypes of the InsP3-R resulting fr om RNA-splicing that are expressed in a tissue-specific and developmen tally specific manner and also resulting from different genes. It is b elieved that there are two Ca2+ release mechanisms, InsP3-induced Ca2 release (IICR) and Ca2+-induced Ca2+ release (CICR). Eggs are good ma terials to analyse the machanism of Ca2+ signalling: fertilized hamste r eggs exhibit repetitive Ca2+ transients as well as the Ca2+ wave. A monoclonal antibody to the InsP3-R inhibited both IICR and CICR respec tively upon injection of InsP3 and Ca2+ into hamster eggs. The antibod y completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. T he results indicate that Ca2+ release in fertilized hamster eggs is me diated solely by the InsP3-R, and that Ca2+-sensitized IICR, but not C ICR, generates Ca2+ waves and Ca2+ oscillations.