Cryo-EM insights into isoform-specific properties of the IP3R2 channel.

Calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs) is a fundamental signaling mechanism that regulates diverse cellular processes. Among the three mammalian IP3R isoforms, IP3R2 is widely expressed, yet its structural basis for activation and regulation remains unclear. Here, we report cryo-EM structures of mammalian IP3R2 in ligand-free (closed) and Ca2+/IP3/ATP-bound (activated) states at 3.3 Å and 3.6 Å resolution, respectively. These structures define the architecture of IP3R2 and reveal conformational transitions associated with channel activation. Although the IP3-binding pocket is conserved, subtype-specific differences in IP3 affinity likely arise from conformational dynamics of the regulatory ARM2 domain. Comparative analyses of IP3R isoforms identify subtype-specific allosteric networks and domain motions that underlie differential regulation. We further define the ATP-binding site and, through mutagenesis and electrophysiology, establish the structural basis for ATP modulation of channel activity. Together, these findings reveal mechanisms of IP3R2 activation and subtype-specific regulation, providing a framework for understanding isoform-dependent Ca2+ signaling.