Structural titration reveals Ca²⁺-dependent conformational landscape of the IP₃ receptor

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are endoplasmic reticulum Ca²⁺ channels whose biphasic dependence on cytosolic Ca²⁺ gives rise to Ca²⁺ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP₃R-mediated Ca²⁺ responses, the structural underpinnings of the biphasic Ca²⁺ dependence that underlies Ca²⁺ oscillations are incompletely understood. Here, we collect cryo-EM images of an IP₃R with Ca²⁺ concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca²⁺ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for relative conformational free energy, we demonstrate that Ca²⁺ binding at a high-affinity site allows IP₃Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca²⁺ concentrations, IP₃Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca²⁺ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca²⁺-dependence of IP₃R channel activity.