Cryo-EM structure of type 1 IP3R channel in a lipid bilayer

Abstract

Type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) is the predominant Ca²⁺-release channel in neurons. IP₃R1 mediates Ca²⁺ release from the endoplasmic reticulum into the cytosol and thereby is involved in many physiological processes. Here, we present the cryo-EM structures of full-length rat IP₃R1 reconstituted in lipid nanodisc and detergent solubilized in the presence of phosphatidylcholine determined in ligand-free, closed states by single-particle electron cryo-microscopy. Notably, both structures exhibit the well-established IP₃R1 protein fold and reveal a nearly complete representation of lipids with similar locations of ordered lipids bound to the transmembrane domains. The lipid-bound structures show improved features that enabled us to unambiguously build atomic models of IP₃R1 including two membrane associated helices that were not previously resolved in the TM region. Our findings suggest conserved locations of protein-bound lipids among homotetrameric ion channels that are critical for their structural and functional integrity despite the diversity of structural mechanisms for their gating.

Fig. 1. Overview of lipids in the IP₃R1-ND structure.

a The cryo-EM density map of the IP₃R1-ND complex is viewed along the membrane plane. Subunits are color-coded and lipid-nanodisc densities are colored gray. b The atomic model of IP₃R1-ND viewed along the membrane plane. A slice through the nanodisc densities (gray mesh) reveals the protein-bound lipid densities (orange). The model of IP₃R1 is shown in wire representation and color-coded by subunit. c TM region of IP₃R1 in nanodiscs viewed from the lumen along the channel’s four-fold symmetry axis. d TM region viewed along the membrane plane; the lipid densities are shown in orange and displayed at 5σ threshold. e Zoomed-in view of lipid densities in inter- and intra-subunit crevices viewed along the four-fold axis from the lumen (left) and cytosol (right). The side-chain of F2586 that constitutes the gate in TM6 is displayed.

Fig. 2. The ion-permeation pathway of IP₃R1 in nanodisc.

a Solvent-accessible pathway along the IP₃R1-ND pore mapped using the program HOLE. A series of residues within the ion-conduction pathway are labeled. Dashed line box indicates the zoomed-in region in c. b Comparison of the pore dimensions for IP₃R1-ND (pink), IP₃R1-LMNG (purple), and IP₃R1-CHAPS (PDB ID: 6MU2; dashed gray line). c A wire representation of the SF in IP₃R1-ND; two opposing subunits are viewed along the membrane plane; narrowest distances between Cα atoms (G2546) or side-chain atoms along the SF are indicated. d The surface electrostatic potential along the ion-permeation pathway. The left panel shows a slice through the channel pore along the four-fold axis with the location of slices perpendicular to the four-fold axis (right panels) indicated by dotted lines. Panels on the right show slices through the pore at E2470, D2551, G2546, and R2597 viewed from the cytosolic side along the symmetry axis.

Fig. 3. Protein–lipid interactions identified in IP₃R1-ND structure.

a IP₃R1-ND cryo-EM density map for TM1–TM6 helices of two opposing subunits; the model is depicted as a ribbon and viewed parallel to the membrane plane. Lipids are represented as orange ball-and-stick models and labeled L1–L7. b TMD densities for one subunit are overlaid with the IP₃R1-ND model viewed parallel to the membrane plane and rotated ~90° from the position in a. c Zoomed-in views of seven lipid-binding sites (L1–L7) identified in the TM region. Lipids are putatively modeled as phosphatidylcholine (see “Methods” section); the PC models are colored by elements and fit into corresponding cryo-EM densities displayed at 3–5σ cut-off values (gray) and 2–2.5σ (orange mesh). TM helices are depicted as ribbons colored by channel subunit. Residues within 5 Å of the lipid molecules are displayed in a stick representation and labeled.

Fig. 4. IP₃R1 domains at the cytosolic-lipid bilayer interface.

a The ILD (light-green) and LNK domain (orange) form a metastable nexus connecting the CY (gray) and TM (purple) regions in the tetrameric IP₃R1 channel; viewed along the membrane plane. The right panel shows a zoomed-in view of the domains (indicated by the gray dashed line in the left panel) at the cytosolic-membrane interface viewed from cytosol along the four-fold axis. b The interface between ILD and the LNK/TM6 of neighboring subunits overlapped with corresponding EM densities (displayed at 4σ). c Close-up view of Zn²⁺ binding site in LNK domain; residues in the C2H2-like Zn²⁺ finger are labeled; the density corresponding to Zn²⁺ is depicted as magenta mesh, contoured at 15σ.

Fig. 5. Model of disease-associated mutations in TM domains of IP₃R1.

a TM4–TM6 helices (gray, wire representation) from two opposing subunits of IP₃R1-ND are shown in side view; lipid molecules (orange) are depicted as ball-and-stick models overlapped with corresponding surface representations (mesh). Residues in the rat IP₃R1 sequence that correspond to the human IP₃R1 mutations associated with Gillespie Syndrome, microcephaly with pontine and cerebellar hypoplasia, spinocerebellar ataxias, and pontocerebellar hypoplasia are depicted as spheres and colored magenta, pink, blue, and yellow, respectively. b TM4–TM6 helices of four subunits with lipids viewed from the cytosol (left panel) and lumen (right panel) perpendicular to the membrane plane.