| Abstract |
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 IP(3)R (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 Ca(2+)-stores but also in cells with depleted Ca(2+) stores. Thapsigargin (ThG) and ionomycin failed to mimic the ATP-induced cluster formation despite the continuous elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Application of IP(3) caused GFP-IP(3)R3 clustering in permeabilized cells, and the response was completely inhibited by heparin, a competitive inhibitor of IP(3)R. Experiments using LIBRAv, an IP(3) biosensor, showed that ATP significantly stimulated IP(3) 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 Ca(2+). When permeabilized cells were stimulated with the threshold of IP(3), the GFP-IP(3)R3 clustering clearly occurred in Ca(2+)-free medium but not in Ca(2+)-containing medium. These results strongly support the hypothesis that the agonist-induced clustering of IP(3)R is triggered by IP(3) binding, rather than [Ca(2+)](i) elevation. Although depletion of the Ca(2+) store by itself does not cause the clustering, it may increase the sensitivity of IP(3)R to cluster formation, leading to facilitation of IP(3)-triggered clustering.
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