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

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) 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 IP(3)Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca(2+ )concentrations, IP3Rs 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 Ca2+-dependence of IP3R channel activity.