The clustering of inositol 1,4,5-trisphosphate (IP(3)) receptors is triggered by IP(3) binding and facilitated by depletion of the Ca(2+) 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 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.
Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan. tojyo@hoku-iryo-u.ac.jp
Yosuke
Y
Tojyo
Takao
T
Morita
Akihiro
A
Nezu
Akihiko
A
Tanimura
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002522:Chlorocebus aethiops; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*
D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium