Characterization of a rapidly dissociating inositol 1,4,5-trisphosphate-binding site in liver membranes.

The binding of inositol 1,4,5-trisphosphate (InsP3) to a specific receptor induces the release of Ca2+ from an intracellular store. In the liver, the KD of a low affinity state of the receptor (RL) found at low Ca2+ concentration ([Ca2+]) is in close agreement with the EC50 of the InsP3-induced Ca2+ release. We have developed an experimental procedure for measuring the rate of dissociation of this low affinity [32P]InsP3-receptor complex in less than 1 s. When the receptor was in the RL state, two kinetic components, RL1 and RL2, were identified with respective rate constants (k(off)) of 1-2 s-1 and 0.03-0.06 s-1. Increasing the [Ca2+] up to 1 microM transformed the receptor into the high affinity state (RH) and decreased the dissociation rate constant to 2 x 10(-2) min-1. We also investigated the time course of the transformation of the receptor from the high affinity (RH) to the low affinity state (RL) after decreasing the [Ca2+] to less than 10 nM. This reversion was dramatically dependent on temperature: at 4 degrees C, the receptor was locked in the RH state, whereas at 37 degrees C the receptor reverted to the RL state with a half-time of less than 1 s. The reversion from the RH state to the RL one is associated to a recovery of InsP3-induced 45Ca2+ release on permeabilized hepatocytes. The rapid and reversible transformation of the InsP3 receptor from an active to an inactive state may be a key event in the Ca2+ release process in intact cells.