Cluster formation of inositol 1,4,5-trisphosphate receptor requires its transition to open state

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP ₃R) Ca²⁺ channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP ₃R forms clusters on the endoplasmic reticulum when cytosolic Ca ²⁺ 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 IP ₃-generating agonists, did not correlate with [Ca²⁺] _C but seemed compatible with cytoplasmic IP₃ concentration. IP₃ production alone induced GFP-IP ₃R1clustering in the absence of a significant increase in [Ca ²⁺]_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 IP ₃-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.