Dynamic regulation of IP3 receptor clustering and activity by IP3.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular Ca(2+) channels. Their regulation by both IP(3) and Ca(2+) allows interactions between IP(3)Rs to generate a hierarchy of intracellular Ca(2+) release events. These can progress from openings of single IP(3)R, through near-synchronous opening of a few IP(3)Rs within a cluster to much larger signals that give rise to regenerative Ca(2+) waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP(3)R3 and shown that low concentrations of IP(3) rapidly and reversibly cause IP(3)Rs to assemble into small clusters. In addition to bringing IP(3)Rs close enough to allow Ca(2+) released by one IP(3)R to regulate the activity of its neighbors; clustering also retunes the regulation of IP(3)Rs by IP(3) and Ca(2+). At resting cytosolic [Ca(2+)], lone IP(3)R are more sensitive to IP(3) and the mean channel open time (approximately 10 ms) is twice as long as for clustered IP(3)R. When the cytosolic free [Ca(2+)] is increased to 1 microM, to mimic the conditions that might prevail when an IP(3)R within a cluster opens, clustered IP(3)R are no longer inhibited and their gating becomes coupled. IP(3), by dynamically regulating IP(3)R clustering, both positions IP(3)R for optimal interactions between them and it serves to exaggerate the effects of Ca(2+) within a cluster. During the course of these studies, we have observed that nuclear IP(3)R stably express one of two single channel K(+) conductances (gamma(K) approximately 120 or 200 pS). Here we demonstrate that for both states of the IP(3)R, the effects of IP(3) on clustering are indistinguishable. These observations reinforce our conclusion that IP(3) dynamically regulates assembly of IP(3)Rs into clusters that underlie the hierarchical recruitment of elementary Ca(2+) release events.