Inhibition of nuclear vesicle fusion by antibodies that block activation of inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate (IP₃) receptors are ligand-gated channels that release intracellular Ca²⁺ stores in response to the second messenger, IP₃. We investigated the potential role of IP₃ receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca²⁺ mobilization is required for nuclear vesicle fusion and implicated IP₃ receptor activity. To test the involvement of IP₃ receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type I IP₃ receptor. Pretreatment of membranes with two of the antibodies inhibited IP₃-stimulated Ca²⁺ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the 'coupling' domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP₃. Because nuclear vesicle fusion was inhibited by antibodies that block Ca²⁺ flux, but not by control and preimmune antibodies, we concluded that the activation of IP₃ receptors is required for fusion. The signal that activates the channel during fusion is unknown.