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

Abstract

The versatility of Ca²⁺ signals derives from their spatio-temporal organization^1,2. 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⁺ waves^1,5,6,7,8,9. 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⁺ signals^3,5.