Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Their regulation by both IP3 and Ca 2+ allows interactions between IP3Rs to generate a hierarchy of intracellular Ca2+ release events. These can progress from openings of single IP3R, through near-synchronous opening of a few IP3Rs within a cluster to much larger signals that give rise to regenerative Ca2+ waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP3R3 and shown that low concentrations of IP3 rapidly and reversibly cause IP3Rs to assemble into small clusters. In addition to bringing IP3Rs close enough to allow Ca2+ released by one IP3R to regulate the activity of its neighbors; clustering also retunes the regulation of IP3Rs by IP3 and Ca2+. At resting cytosolic [Ca2+], lone IP3R are more sensitive to IP3 and the mean channel open time (~10 ms) is twice as long as for clustered IP3R. When the cytosolic free [Ca 2+] is increased to 1 μM, to mimic the conditions that might prevail when an IP3R within a cluster opens, clustered IP 3R are no longer inhibited and their gating becomes coupled. IP 3, by dynamically regulating IP3R clustering, both positions IP3R for optimal interactions between them and it serves to exaggerate the effects of Ca2+ within a cluster. During the course of these studies, we have observed that nuclear IP3R stably express one of two single channel K+ conductances (γK ~20 or 200 pS). Here we demonstrate that for both states of the IP3R, the effects of IP3 on clustering are indistinguishable. These observations reinforce our conclusion that IP3 dynamically regulates assembly of IP3Rs into clusters that underlie the hierarchical recruitment of elementary Ca2+ release events. © 2009 Landes Bioscience.
