Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+

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Abstract
Inositol trisphosphate (InsP3) is a second messenger that triggers release of Ca2+ from intracellular stores through its cognate receptor — a ligand-gated ion channel. This paper shows that InsP3 receptors aggregate in response to InsP3, resulting in a lower probability of channel opening, and that increases in intracellular Ca2+ can effectively relieve this inhibition and enhance the coupling between receptors in a cluster. This mechanism may serve to enhance the sensitivity to changes in intracellular Ca2+. This papers shows that inositol-1,4,5-trisphosphate (InsP3) receptors aggregate in responses to InsP3, resulting in a lower probability of channel opening, and that increases in intracellular Ca2+ can effectively relieve this inhibition and enhance the coupling between receptors in a cluster. This mechanism probably serves to enhance the sensitivity to changes in intracellular Ca2+. The versatility of Ca2+ signals derives from their spatio-temporal organization1,2. For Ca2+ signals initiated by inositol-1,4,5-trisphosphate (InsP3), this requires local interactions between InsP3 receptors (InsP3Rs)3,4 mediated by their rapid stimulation and slower inhibition4 by cytosolic Ca2+. This allows hierarchical recruitment of Ca2+ release events as the InsP3 concentration increases5. Single InsP3Rs respond first, then clustered InsP3Rs open together giving a local ‘Ca2+ puff’, and as puffs become more frequent they ignite regenerative Ca2+ waves1,5,6,7,8,9. Using nuclear patch-clamp recording10, here we demonstrate that InsP3Rs are initially randomly distributed with an estimated separation of ∼1 μm. Low concentrations of InsP3 cause InsP3Rs to aggregate rapidly and reversibly into small clusters of about four closely associated InsP3Rs. At resting cytosolic [Ca2+], clustered InsP3Rs open independently, but with lower open probability, shorter open time, and less InsP3 sensitivity than lone InsP3Rs. Increasing cytosolic [Ca2+] reverses the inhibition caused by clustering, InsP3R gating becomes coupled, and the duration of multiple openings is prolonged. Clustering both exposes InsP3Rs to local Ca2+ rises and increases the effects of Ca2+. Dynamic regulation of clustering by InsP3 retunes InsP3R sensitivity to InsP3 and Ca2+, facilitating hierarchical recruitment of the elementary events that underlie all InsP3-evoked Ca2+ signals3,5.