Multiple mechanisms of regulation of the inositol 1,4,5-trisphosphate receptor by calcium.

Ca2+ mobilisation by inositol 1,4,5-trisphosphate (InsP3) is a complex phenomenon which involves positive and negative feedback regulation by cytosolic Ca2+. It has been shown that Ca2+ increased the affinity of [3H]-InsP3 binding to liver membranes and inhibited [3H]-InsP3 binding to cerebellar membranes. We investigated the effects of Ca2+ on the [3H]-InsP3 binding to receptor solubilised and rapidly purified by immunoprecipitation. The InsP3 binding to the purified liver receptor was insensitive to the addition of Ca2+, indicating that Ca2+ did not interact directly with the receptor. The loss of the Ca2+ effect on liver receptor affinity was reproduced by alkaline treatment of liver membranes, which is known to extract the peripheral membrane proteins. This suggests that Ca2+ regulates the liver InsP3 receptor by interacting with a membrane-associated protein. Ca2+ inhibited the binding of [3H]-InsP3 to purified cerebellar receptors as was found with the membrane fraction. The treatment of the purified cerebellar receptor with media of high ionic strength or at alkaline pH did not abolish the effect of Ca2+ on the receptor. This indicates that the inhibitory effect of Ca2+ on [3H]-InsP3 binding to cerebellar membranes occurs either via direct interaction with the receptor or via an integral protein strongly associated with the receptor. In conclusion, the mechanisms of regulation of InsP3-induced Ca2+ release by Ca2+ involve different molecular support in cerebellum and in liver. This may reflect different regulation dependent on the receptor type.