Structure of IP3R channel: high-resolution insights from cryo-EM

Abstract

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ubiquitously expressed intracellular Ca²⁺ channels and the major mediators of cellular Ca²⁺ signals generated by the release of Ca²⁺ ions from intracellular stores in response to a variety of extracellular stimuli. Despite established physiological significance and proven involvements of IP₃R channels in many human diseases, detailed structural basis for signal detection by these ion channels and their gating remain obscure. Recently, single particle electron cryomicroscopy (cryo-EM) has yielded a long-awaited near-atomic resolution structure of the entire full-length type 1 IP₃R. This structure provided exciting mechanistic insights into the molecular assembly of IP₃R, revealing the pronounced structural conservation of Ca²⁺ release channels and raising many fundamental and controversial questions on their activation and gating. Here we summarize the major technological advances that propelled our cryo-EM analysis of IP₃R to near-atomic resolution and discuss what the future holds for structural biology of Ca²⁺ release channels.

Figure 1. Resolution assessment and structure validation

Two 3D reconstructions of IP₃R1 were performed entirely independently with Relion1.3 (a) and EMAN 2.1 (b) software packages using the same cryo-EM data. (c) Shown are the gold-standard FSC curves [41,43] for the final 3D reconstructions generated with Relion1.3 (black line) and EMAN2.1 (green line). (d) The FSC profiles for the Relion1.3 reconstructions: the blue line shows the FSC curve computed for the data in which the phases were randomized from 6 Å to Nyquist; the gold standard FSC curve is shown with a black line and is nearly identical to the “true FSC” (red line) calculated as described in [48].

Figure 2. Structure of rat cerebellar IP₃R1 determined by single-particle cryo-EM

Orthogonal views for the IP₃R1 channel: (a) view along the membrane plane; the detergent-phospholipid belt is observed at low density threshold (semi-transparent gray mesh) and denotes the position of the channel complex in the membrane with the cytosolic domains facing up; (b) view down the four-fold axis from the cytosolic side. (c) Topological scheme of the ten domains identified in the individual IP₃R1 subunit.

Figure 3. Structural conservation of ion channels. (a)

Structures of IP₃R1 (3jav), RyR1 (5tb0) and TRPA1 (3j9p) viewed along the membrane plane. (b) Correspondence of domains between IP₃R1 and RyR1 based on their structural alignments. Reported are Cα RMSDs for the aligned residue pairs. Structure comparison was performed on the entire domains with the following exceptions: ARM1 is aligned with NSol domains and partial JSol domain (residues 1657–1720), the remaining portion of the JSol domain (residues 1721–2144) is aligned with HD, and BSol residues 2145–2599 are aligned with residues 1119–1494 from ARM2. Structurally conserved domains are color-coded based on IP₃R1.

Figure 4. Structural conservation of putative Ca²⁺ sensor region. (a)

The armadillo repeat domain 3 (ARM3) of IP₃R1 (3jav) and the C-solenoid of RyR1 (5tb0) share structural and sequence conservation. IP₃R1 is colored chartreuse and RyR1 is colored tan. Cα RMSD is 1.8 Å. Dashed line indicates Ca²⁺ binding pocket identified in RyR1 [25]. The conserved residues E2101 (IP₃R1) and E4032 (RyR1) that have been implicated in channel Ca²⁺ sensitivity are colored red (a–c). (b) Two zoomed-in views of the RyR1 Ca²⁺ binding pocket (left panels) and the structurally equivalent residues of IP₃R1 (middle panels) are shown. Right panels show the overlay of RyR1 and IP₃R1 putative Ca²⁺ binding pockets with all identical residues colored blue. (c) Sequence alignment based on the structural overlap for the ARM3 and C-solenoid domains is shown. Identical residues are colored blue and structurally equivalent residues in IP₃R1 based on the putatively identified RyR1 Ca²⁺ binding pocket are indicated with a box.