Clustering of IP(3) receptors by IP(3) retunes their regulation by IP(3) and Ca(2+)

The versatility of Ca(2+) signals derives from their spatio-temporal organization1,2. For Ca(2+) signals initiated by inositol trisphosphate (IP(3)) this requires local interactions between IP(3) receptors (IP(3)R)3,4 mediated by their rapid stimulation and slower inhibition4 by cytosolic Ca(2+). This allows hierarchical recruitment of Ca(2+) release events as the IP(3) concentration increases5. Single IP(3)R respond first, then clustered IP(3)R open together giving a local Ca(2+) puff, and as puffs become more frequent they ignite regenerative Ca(2+) waves1,5-9. We demonstrate, using nuclear patch-clamp recording10, that IP(3)R are initially randomly distributed with an estimated separation of ~1 μm. Low concentrations of IP(3) cause IP(3)R to aggregate rapidly and reversibly into small clusters of ~4 closely associated IP(3)R. At resting cytosolic [Ca(2+)], clustered IP(3)R open independently, but with lower open probability (P(o)), shorter open time, and lesser IP(3) sensitivity than lone IP(3)R. Increasing cytosolic [Ca(2+)] reverses the inhibition caused by clustering, IP(3)R gating becomes coupled, and the duration of multiple openings is prolonged. Clustering both exposes IP(3)R to local Ca(2+) rises and increases the effects of Ca(2+). Dynamic regulation of clustering by IP(3) tunes IP(3)R sensitivity to IP(3) and Ca(2+), facilitating hierarchical recruitment of the elementary events that underlie all IP(3)-evoked Ca(2+) signals3,5.