The clustering of inositol 1,4,5-trisphosphate (IP3) receptors is triggered by IP3 binding and facilitated by depletion of the Ca2+ store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) form clusters following agonist stimulation, but its mechanism remains controversial. In this study, we visualized the clustering of green fluorescent protein (GFP)-tagged type 3 IP3R (GFP-IP(3)R3) in cultured living cells using confocal microscopy. Stimulation with ATP evoked GFP-IP(3)R3 clustering not only in cells with replete Ca2+-stores but also in cells with depleted Ca2+ stores. Thapsigargin (ThG) and ionomycin failed to mimic the ATP-induced cluster formation despite the continuous elevation of intracellular Ca2+ concentration ([Ca2+](i)). Application of IP3 caused GFP-IP(3)R3 clustering in permeabilized cells, and the response was completely inhibited by heparin, a competitive inhibitor of IP3R. Experiments using LIBRAv, an IP3 blosensor, showed that ATP significantly stimulated IP3 generation even in store-depleted cells. We also found that pretreatment with ThG accelerated or enhanced the ATP-induced clustering in both the presence and absence of extracellular Ca2+. When permeabilized cells were stimulated with the threshold of IP3, the GFP-IP3R3 clustering clearly occurred in Ca2+-free medium but not in Ca2+-containing medium. These results strongly support the hypothesis that the agonist-induced clustering of IP3R is triggered by IP3 binding, rather than [Ca2+]i elevation. Although depletion of the Ca2+ store by itself does not cause the clustering, it may increase the sensitivity of IP3R to cluster formation, leading to facilitation of IP3-triggered clustering.