Structural titration reveals Ca-dependent conformational landscape of the IP receptor.

Inositol 1,4,5-trisphosphate receptors (IPRs) are endoplasmic reticulum Ca channels whose biphasic dependence on cytosolic Ca gives rise to Ca oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IPR-mediated Ca responses, the structural underpinnings of the biphasic Ca dependence that underlies Ca oscillations are incompletely understood. Here, we collect cryo-EM images of an IPR with Ca concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca 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 Ca binding at a high-affinity site allows IPRs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca concentrations, IPRs preferentially enter an inhibited state stabilized by a second, low-affinity Ca binding site. Together, these studies provide a mechanistic basis for the biphasic Ca-dependence of IPR channel activity.