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

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are endoplasmic reticulum Ca(2+) channels whose biphasic dependence on cytosolic Ca(2+) gives rise to Ca(2+) oscillations that regulate fertilization, cell division and cell death. Despite the critical roles of IP(3)R-mediated Ca(2+) responses, the structural underpinnings of the biphasic Ca(2+) dependence that underlies Ca(2+) oscillations are incompletely understood. Here, we collect cryo-EM images of an IP(3)R with Ca(2+) concentrations spanning five orders of magnitude. Unbiased image analysis reveals 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 relative conformational 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+) concentrations, 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.