Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP3R forms clusters on the endoplasmic reticulum when cytosolic Ca2+ concentration ([Ca2+]C) is elevated. However, the molecular mechanism of IP3R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP3R type 1 (GFP-IP3R1), evoked by IP3-generating agonists, did not correlate with [Ca2+]C but seemed compatible with cytoplasmic IP3 concentration. IP3 production alone induced GFP-IP3R1 clustering in the absence of a significant increase in [Ca2+]C but elevated [Ca2+]C without IP3 production did not. Moreover IP3R1 mutants that do not undergo an IP3-induced conformational change failed to form clusters. Thus, IP3R clustering is induced by its IP3-induced conformational change to the open state. We also found that GFP-IP3R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.