Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3

Abstract

Inositol trisphosphate receptors (IP₃Rs) are ubiquitous Ca²⁺-permeable channels that mediate release of Ca²⁺ from the endoplasmic reticulum, thereby regulating numerous processes including cell division, cell death, differentiation and fertilization. IP₃Rs are jointly activated by inositol trisphosphate (IP₃) and their permeant ion, Ca²⁺. At high concentrations, however, Ca²⁺ inhibits activity, ensuring precise spatiotemporal control over intracellular Ca²⁺. Despite extensive characterization of IP₃R, the mechanisms through which these molecules control channel gating have remained elusive. Here, we present structures of full-length human type 3 IP₃Rs in ligand-bound and ligand-free states. Multiple IP₃-bound structures demonstrate that the large cytoplasmic domain provides a platform for propagation of long-range conformational changes to the ion-conduction gate. Structures in the presence of Ca²⁺ reveal two Ca²⁺-binding sites that induce the disruption of numerous interactions between subunits, thereby inhibiting IP₃R. These structures thus provide a mechanistic basis for beginning to understand the regulation of IP₃R.

Figure 1. Structure of human type 3 IP₃R in a ligand-free state

a, Structure of hIP₃R3 viewed in the plane of the membrane with the cytoplasmic domain at the top. A single subunit is colored according to domain with BTF1 in purple, BTF2 in blue, ARM1 in violet, the CLD in cyan, ARM2 in green, ARM3 in yellow, the JD in orange and the TMD in red. Gray lines represent the approximate position of the membrane. b, Structure of the cytoplasmic domain viewed from the cytoplasm. A single subunit is colored according to domain. ARM3 is removed for clarity. c, Structure of the transmembrane domain viewed from the cytoplasm. S1–S4 helices are colored red, S1′ and S1″ blue and S5, S6 and pore helix green. d–e, Structure of the ion conduction pathway viewed in the plane of the membrane and plot of pore radius. Residues comprising the luminal vestibule, the selectivity filter and S6 gate are highlighted. Front and rear subunits are removed for clarity.

Figure 2. IP₃ binding site in two IP₃-bound conformations

a, d, Superposition of (a) IP₃ class 1 (colored by domain) and apo (grey) or (d) IP₃ class 2 (colored by domain) and apo (grey) viewed from in the plane of the membrane (left) and the cytoplasm (right). b, e, Superposition of IP₃-binding domain of (b) IP₃ class 1 (colored by domain) and apo (grey) or (e) IP₃ class 2 (colored by domain) and apo (grey) aligned by BTF1 and BTF2. IP₃ and the side chains of IP₃-coordinating residues are shown as sticks. c, f, Superposition of ARM3, the JD and S6 of (c) IP₃ class 1 (colored by domain) and apo (grey) or (f) IP₃ class 2 (colored by domain) and apo (grey) aligned by the TMD and viewed from the lumen.

Figure 3. Ensemble of IP₃-bound conformations

a, Structures of IP₃ class 1, IP₃ class 32, IP₃ class 4, IP₃ class 5 and IP₃ class 2 colored by domain. The ARM2 domain of subunits marked with an * adopt a class 2-like conformation. b, Structures of IP₃ class 1, IP₃ class 3, IP₃ class 4, IP₃ class 5 and IP₃ class 2. Domains adopting class 1-like domains are colored orange and domains adopting class 2-like conformations are colored cyan.

Figure 4. Ca²⁺ binding sites in Ca²⁺-bound hIP₃R3

a, Monomeric structure of Ca²⁺-bound hIP₃R3 colored by domain viewed in the plane of the membrane. Ca²⁺ and Zn²⁺ ions are shown as spheres. b, CD Ca²⁺-binding site at the CLD-ARM2 interface. Residues coordinating the Ca²⁺ are shown as sticks and the CD Ca²⁺ ion is shown as a magenta sphere. c, Superposition of the CLD and ARM2 of Ca²⁺-bound (colored by domain) and apo (grey, left) or Ca²⁺-bound (colored by domain) and IP₃ class 2 (grey, right) aligned by the CLD. d, JD Ca²⁺-binding site at the ARM3-JD interface. Residues coordinating the Ca²⁺ are shown as sticks and the Ca²⁺ ion is shown as a green sphere. e, Superposition of ARM3 and the JD of Ca²⁺-bound (colored by domain) and apo (grey) aligned by the JD.

Figure 5. Ca²⁺ binding disrupts CD intra- and inter-subunit interactions

a–b, Cytoplasmic domain of (a) apo and (b) Ca²⁺-bound colored by domain, viewed from the cytoplasm. Spheres represent the Cα position of Trp168 of BTF1 and Lys426 of BTF2 (pink) and Pro140 of BTF1 and Ala1291 of ARM2 (brown). c–d, Structure of (c) apo and (d) Ca²⁺-bound colored by domain, viewed in the plane of the membrane. Front and rear subunits are removed for clarity. Ca²⁺ and Zn²⁺ ions are shown as spheres. Grey lines represent the approximate position of the membrane.

Figure 6. IP₃-induced conformational changes

a, High IP₃-Ca²⁺ structure colored by distance of Cα deviation from the Ca²⁺-bound structure viewed in the plane of the membrane. b, IP₃ class 2 colored by distance of Cα deviation from the apo structure viewed in the plane of the membrane. c, Cytoplasmic domain of high IP₃-Ca²⁺ structure colored by distance of Cα deviation from the Ca²⁺-bound structure viewed from the cytoplasm. d, Cytoplasmic domain of IP₃ class 2 colored by distance of Cα deviation from the apo structure viewed from the cytoplasm. IP₃ and Ca²⁺ ions are shown as spheres.

Figure 7. Model for IP₃R regulation by IP₃ and Ca²⁺

In the absence of Ca²⁺ and IP₃, IP₃R adopts a closed state. In the presence of IP₃, IP₃R adopts one of an ensemble of pre-activated states that are in equilibrium. In the presence of high Ca²⁺, the BTF ring is dissociated and the channel adopts an inhibited state. In the presence of high Ca²⁺ and IP₃, the BTF ring is dissociated and the channel adopts an inhibited state. In the presence of low Ca²⁺ and IP₃, the channel adopts a hypothetical activated state in which the S6 gate is opened.