Structural titration reveals Ca ²⁺ -dependent conformational landscape of the IP ₃ receptor

Inositol 1,4,5-trisphosphate receptors (IP ₃ Rs) are intracellular Ca ²⁺ -permeable cation channels whose biphasic dependence on cytoplasmic Ca ²⁺ gives rise to cytosolic Ca ²⁺ oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP ₃ R-mediated Ca ²⁺ oscillations, the structural underpinnings of the biphasic Ca ²⁺ dependence that underlies Ca ²⁺ oscillations are incompletely understood. Here, we collected images of an IP ₃ R with Ca ²⁺ at concentrations spanning five orders of magnitude. Unbiased image analysis revealed that Ca ²⁺ 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 free energy, we demonstrate that Ca ²⁺ binding at a high-affinity site allows IP ₃ Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca ²⁺ , IP ₃ Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca ²⁺ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca ²⁺ -dependence of IP ₃ R channel activity.