Analysis of IP3 Receptor Activation Using Novel Partial Agonists

Abstract

Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2+ channels. Each subunit of the tetrameric IP3R comprises a N-terminal IP3-binding site, a cytosolic domain and six transmembrane domains (TMD) near the C-terminal. TMD5 and 6 form the pore. IP3 binding to the IP3-binding core (IBC, residues 224-604) initiates conformational changes that lead to opening of the pore. The mechanisms are unresolved but require the suppressor domain (SD, residues 1-223) and interactions between N and C termini. We have synthesized a family of 2-O-modified IP3 analogues that are partial agonists of the IP3R. In defining their properties, we identify a novel form of partial agonism that allows us to define key steps in IP3R activation. By combining analysis of ΔG for ligand binding, single-channel recordings and molecular modelling we show that these partial agonists are IP3-like in their interactions with the IBC, but they less effectively rearrange its relationship with the SD. The partial agonists open the channel at slower rates than full agonists. IP3R with a point mutation in the SD that occupies a position similar to the 2-O-substituent of the partial agonists has reduced open probability that is similar for full and partial agonists. This suggests that bulky or charged substituents at the IBC-SD interface, whether provided by the ligand or SD, frustrate an obligatory coupling between IBC and SD. Our analysis of ΔG for ligand binding to truncated IP3R shows that conformational changes initiated by IP3 binding propagate entirely via the SD to the pore. We suggest that IP3 first closes the clamp-like structure of the IBC, then the IBC interacts with the SD to give a compact structure that allows the SD alone to signal onwards to the pore.