Structure and Function of IP3 Receptors

Abstract

Inositol 1,4,5-trisphosphate receptors (IP₃Rs), by releasing Ca²⁺ from the endoplasmic reticulum (ER) of animal cells, allow Ca²⁺ to be redistributed from the ER to the cytosol or other organelles, and they initiate store-operated Ca²⁺ entry (SOCE). For all three IP₃R subtypes, binding of IP₃ primes them to bind Ca²⁺, which then triggers channel opening. We are now close to understanding the structural basis of IP₃R activation. Ca²⁺-induced Ca²⁺ release regulated by IP₃ allows IP₃Rs to regeneratively propagate Ca²⁺ signals. The smallest of these regenerative events is a Ca²⁺ puff, which arises from the nearly simultaneous opening of a small cluster of IP₃Rs. Ca²⁺ puffs are the basic building blocks for all IP₃-evoked Ca²⁺ signals, but only some IP₃ clusters, namely those parked alongside the ER-plasma membrane junctions where SOCE occurs, are licensed to respond. The location of these licensed IP₃Rs may allow them to selectively regulate SOCE.

Figure 1.

IP₃ receptors deliver Ca²⁺ to the cytosol and organelles. (A) By releasing Ca²⁺ from the endoplasmic reticulum (ER), IP₃Rs can deliver Ca²⁺ to the cytosol, to other IP₃Rs to ignite regenerative signals, or to the close appositions (membrane contact sites, supported by scaffold proteins) between the ER and other organelles. The latter include mitochondria and lysosomes, which can then accumulate Ca²⁺ via their low-affinity uptake systems from the high local Ca²⁺ concentration provided by IP₃Rs. (B) Loss of Ca²⁺ from the ER also activates STIM1, which then binds to Orai at ER–plasma membrane (PM) junctions to initiate store-operated Ca²⁺ entry (SOCE).

Figure 2.

IP₃ receptors are stimulated by IP₃ and Ca²⁺. (A) Many receptors, including G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), can stimulate phospholipase C (PLC), leading to production of IP₃, which then binds to IP₃Rs in the endoplasmic reticulum (ER). (B) IP₃ binding to IP₃R primes them to bind Ca²⁺, which then stimulates the channel to open, allowing Ca²⁺ to flow out of the ER. (C) This dual regulation of IP₃Rs by IP₃ and Ca²⁺ allows them to mediate regenerative signals propagated by Ca²⁺-induced Ca²⁺ release (CICR). PM, Plasma membrane.

Figure 3.

IP₃ receptor structure. (A) Cryoelectron microscopy (cryo-EM) of IP₃R1 shows its tetrameric mushroom-like structure. (From Fan et al. 2015; adapted, with permission, from Springer Nature © 2015.) Subunits are color-coded. A similar structure has been reported for IP₃R3 (Paknejad and Hite 2018). (B) View from the cytosol. (C) The amino-terminal region of each IP₃R subunit comprises the suppressor domain (SD) with the “hot spot” loop through which it contacts an adjacent subunit; and the IP₃-binding core (IBC), with its α and β domains. The essential 4- and 5-phosphates of IP₃ interact predominantly with residues on the inner surface of the β and α domains, respectively, to trigger partial closure of clam-like IBC. (D) Schematic representation showing a single IP₃R1 subunit, highlighting the IBC, where IP₃ binds, two Ca²⁺-binding sites at interfaces between ARM1 and ARM2 domains, and between the LNK and ARM3 domains (Paknejad and Hite 2018). The α-helical rod (carboxy-terminal domain [CTD]) extending from LNK to the cap of the mushroom was resolved in structures from one laboratory (Fan et al. 2015, 2018), but not in the structures determined by another laboratory (Paknejad and Hite 2018). It is clear that conformational changes initiated by IP₃ binding must pass through a critical nexus formed by the LNK (from the pore region) and intervening lateral domains (ILDs) (from the cytosolic domain). (E) Structures of IP₃ and adenophostin A, showing how the latter has structures equivalent to the essential 4- and 5-phosphates of IP₃.

Figure 4.

IP₃ receptors interact with many accessory proteins. (A) Proteins that interact with IP₃Rs shown according to the regions of the IP₃R with which they interact. (From Prole and Taylor 2016; adapted under the terms of the Creative Commons Attribution License [CC BY, 2016].) AKAP9, A-kinase anchoring protein 9; AKT1, RAC-α serine/threonine protein kinase; BANK1, B-cell scaffold protein with ankyrin repeats; Bcl-2, B-cell lymphoma 2; B₂R, bradykinin B₂ receptor; BRCA1, breast and ovarian cancer susceptibility gene 1; CaBP1, Ca²⁺-binding protein 1; CaM, calmodulin; CARP, carbonic anhydrase-related protein; CDK1, cyclin-dependent kinase 1; CIB1, Ca²⁺- and integrin-binding protein 1; CYB, cyclin-B1; EB3, end-binding protein 3; EGFR, epidermal growth factor receptor; GRP-75, glucose-regulated protein 75; HTT, huntingtin; IRBIT, IP₃-binding protein released with IP₃; mGluR1, metabotropic glutamate receptor 1; NCS-1, neuronal Ca²⁺-sensor 1; PAR-2, protease-activated receptor 2; PKA, protein kinase A; PLC-β, phospholipase Cβ; PLC-γ, phospholipase Cγ, PS-1/PS-2, presenilin 1/2; NCX1, Na⁺/Ca²⁺ exchanger 1; PKC, protein kinase C; PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; STIM1, stromal interaction molecule 1; TGM2, transglutaminase-2; VDAC1, voltage-dependent anion channel 1. Proteins shown in red inhibit the activity of IP₃Rs. Original sources for additional interactions shown here are ataxins (Chen et al. 2008; Liu et al. 2009), CARP (Hirota et al. 2003), and BRCA1 (Hedgepeth et al. 2015). (B) Examples of proteins shown according to whether they facilitate delivery of IP₃ to IP₃Rs, intracellular distribution of IP₃Rs, IP₃R activation or delivery of Ca²⁺ to specific targets.

Figure 5.

Immobile IP₃ receptor clusters initiate Ca²⁺signals. (A) HeLa cells with endogenous IP₃R1 tagged with EGFP showing IP₃R puncta (green) within endoplasmic reticulum (ER) membranes (red). (B) Diffraction-limited image of a punctum recorded using total internal reflection fluorescence (TIRF) microscopy, and the superimposed super-resolution (STORM) image showing IP₃Rs (red) within the punctum. The magenta square indicates the approximate size of a single tetrameric IP₃R. (C) IP₃Rs appear to be diffusively distributed within a punctum, suggesting the need for a scaffold to corral IP₃Rs. (D) TIRF images of a HeLa cell in which the ER has been depleted of Ca²⁺ by treatment with thapsigargin, showing the distribution of STIM1 (red) and IP₃R (green). The enlarged image shows that stromal interaction molecule (STIM) puncta form alongside the ER where the immobile IP₃Rs that are licensed to respond are parked. (Data results for A–D are from Thillaiappan et al. 2017.) (E) Licensed IP₃Rs parked alongside the ER–PM junctions where store-operated Ca²⁺ entry (SOCE) occurs may allow substantial loss of Ca²⁺ from that part of the ER without causing appreciable Ca²⁺ loss from the remaining ER. The ER–PM junction with its associated licensed IP₃Rs may constitute the basic functional unit for SOCE.