Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment (‘ex cellula’), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells have demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way for structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore ∼7 nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide evidence to support the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release.
Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyR) are the two major families of intracellular Ca2+-release channels in animal cells (Fig. 1A). IP3Rs are expressed in most cells, whereas RyRs have a more restricted distribution. RyRs are most abundant in excitable cells, notably in striated muscle, where they contribute to excitation–contraction coupling (Fig. 1A) (Van Petegem, 2014). In this Review, we focus on IP3Rs, and how methods applied to IP3Rs removed from intact cells have contributed to our understanding of IP3R behaviour. Progress in our understanding of IP3Rs and RyRs has advanced in parallel, and with this progress it became clear that the two families share structural and functional features (Baker et al., 2017; Seo et al., 2012). Hence, despite our focus on IP3Rs, we draw also on evidence from analyses of RyRs.
Classic work by Sydney Ringer demonstrated that cardiac muscle contraction requires extracellular Ca2+ (Ringer, 1883). This was, with the benefit of hindsight, the first of many studies to show that the contributions to physiological responses of extracellular Ca2+ and Ca2+ held within intracellular stores are entangled. For cardiac muscle, depolarization of the plasma membrane (PM) causes voltage-gated Ca2+ channels (Cav1.2, also known as CACNA1C) to open, and the local increase in cytosolic free Ca2+ concentration ([Ca2+]c) is then amplified by Ca2+-induced Ca2+ release (CICR) through type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum (Bers, 2002) (Fig. 1A). CICR and the local Ca2+ signalling that is required to avoid CICR from becoming explosive have become recurrent themes in the field of Ca2+ signalling (Rios, 2018). Fluorescent Ca2+ indicators and optical microscopy now allow Ca2+ sparks, local Ca2+ signals evoked by a small cluster of RyRs, to be measured with exquisite subcellular resolution in cardiac muscles (Cheng and Lederer, 2008). However, it was studies of permeabilized cells (‘skinned’ fibres) that provided the first evidence for CICR in muscle (Endo et al., 1970; Fabiato and Fabiato, 1979). Analyses of RyRs that were reconstituted into planar lipid bilayers first showed that RyRs form large-conductance cation channels that are biphasically regulated by cytosolic Ca2+ (Lai et al., 1988; Meissner, 2017). Finally, analyses of RyR fragments by X-ray crystallography (Van Petegem, 2014) and of complete RyRs by cryo-electron microscopy (des Georges et al., 2016; Efremov et al., 2015; Peng et al., 2016; Yan et al., 2015; Zalk et al., 2015) are revealing the structural basis of RyR behaviour.
Progress towards understanding the second major family of intracellular Ca2+-release channels, the IP3Rs, began with an influential review in which a causal link between receptor-stimulated turnover of phosphatidylinositol and Ca2+ signalling was proposed (Michell, 1975). Subsequent work established that many receptors stimulate phospholipases C, which cleave phosphatidylinositol 4,5-bisphosphate to produce IP3 and diacylglycerol (Berridge, 1993) (Fig. 1A). IP3 provides the link to Ca2+ signalling; not, as first envisaged, by directly stimulating Ca2+ entry across the PM (Michell, 1975), but by stimulating Ca2+ release from the endoplasmic reticulum (ER) through IP3Rs (Berridge and Irvine, 1984; Streb et al., 1983). Another influential review suggested the link between IP3-evoked Ca2+ release and Ca2+ entry across the PM, and proposed that loss of Ca2+ from the ER stimulated Ca2+ entry (Putney, 1986). The workings of this store-operated Ca2+ entry (SOCE) pathway are now clear: dissociation of Ca2+ from the luminal EF-hand motif of a protein embedded in the ER membrane, stromal interaction molecule 1 (STIM1), causes STIM1 to oligomerize and expose a cytosolic domain, through which it stimulates opening of a Ca2+-selective channel in the PM (Feske et al., 2006; Prakriya and Lewis, 2015). The Ca2+ channel that mediates SOCE is a hexameric assembly of Orai subunits (Hou et al., 2012; Yen and Lewis, 2018), grandiloquently named from Greek mythology after the keepers of heaven (Feske et al., 2006).
IP3Rs and RyRs are biphasically regulated by cytosolic Ca2+ (Bezprozvanny et al., 1991). For IP3Rs exposed to IP3, a modest increase in [Ca2+]c stimulates opening, whereas a higher [Ca2+]c is inhibitory (Foskett et al., 2007; Iino, 1990). Hence IP3Rs, at least once they have bound IP3 (Alzayady et al., 2016), can, like RyRs, mediate CICR (Fig. 1B,C). As with RyRs, IP3Rs assemble into clusters, within which opening of one IP3R ignites the activity of its neighbours to generate local ‘Ca2+ puffs’ (Fig. 1C) (Smith and Parker, 2009; Thillaiappan et al., 2017), analogous to Ca2+ sparks in muscle. These behaviours illustrate some of the many similarities between IP3Rs and RyRs, which include their close structural relationship (Baker et al., 2017; Seo et al., 2012; Van Petegem, 2014). Although IP3Rs and RyRs are the major intracellular Ca2+-release channels and the focus of this Review, they are not the only intracellular Ca2+ channels. Brief descriptions of additional intracellular Ca2+ channels are provided in Box 1.
IP3Rs and RyRs are the major intracellular Ca2+-release channels in most cells and the major links between extracellular stimuli and Ca2+ release from the ER or sarcoplasmic reticulum (SR) (Fig. 1A), but they are not the only intracellular Ca2+ channels (Taylor et al., 2009). Polycystin-2 (also known as TRPP2 or PKD2), a member of the transient receptor potential (TRP) superfamily, is also expressed in the ER and is activated by Ca2+ (Koulen et al., 2002). A variety of Ca2+-permeable channels are expressed in lysosomes, including those regulated by: luminal pH and ATP (e.g. P2X purinoceptor 4, P2RX4) (Huang et al., 2014), cytosolic nicotinic acid adenine dinucleotide phosphate (NAADP; two pore channel 2, TPC2) (Morgan and Galione, 2013), and the lysosomal membrane lipid, phosphatidylinositol 3,5-bisphosphate (transient receptor potential mucolipin 1 channel, TRPML1) (Cao et al., 2017). The mitochondrial uniporters (MCU) comprise another important family of intracellular Ca2+ channels (Oxenoid et al., 2016; Patron et al., 2013). Opening of MCU is triggered by large local increases in [Ca2+]c, causing Ca2+ to flow rapidly from the cytosol across the inner mitochondrial membrane and into the mitochondrial matrix, where Ca2+ regulates many activities (Rizzuto et al., 2012). A recurrent theme in Ca2+ signalling is the importance of interactions between Ca2+ channels in different membranes: store-operated Ca2+ entry is activated after loss of Ca2+ from the ER through IP3Rs; mitochondrial Ca2+ uptake is driven by local Ca2+ release through IP3Rs and RyRs (Csordas et al., 2018); NAADP-evoked Ca2+ release from lysosomes is amplified by CICR through closely apposed IP3Rs or RyRs (Morgan and Galione, 2013); and Ca2+ puffs and sparks are ignited by CICR triggering Ca2+ release within clusters of IP3Rs or RyRs (Fig. 1C) (Cheng and Lederer, 2008; Rios, 2018; Thillaiappan et al., 2017).
The productive interplay between studies of minimally perturbed tissue, facilitated by a plethora of Ca2+ indicators (Lock et al., 2015), fluorescent proteins (Rodriguez et al., 2017) and fluorescence microscopy techniques (Thorn, 2016), alongside analyses of cellular components, has shaped our understanding of Ca2+ signalling. Here, we consider how analyses of IP3Rs conducted outside their normal intracellular environment (ex cellula) have advanced our understanding of IP3-evoked Ca2+ signals. We begin by considering how analyses of permeabilized cells established that the ER is the major intracellular Ca2+ store and that IP3 releases Ca2+ from it. Radioligand binding analyses then both identified the sites to which IP3 binds to activate IP3Rs, and paved the way to structural studies, which we show are now coming close to revealing how IP3 binding causes the pore of the IP3R to open. We conclude by considering the contributions of electrophysiological recordings to our understanding of IP3R gating.
Lessons from permeabilized cells
Permeabilized cells allow the Ca2+ content of intracellular organelles to be measured under conditions where the intracellular environment can be precisely controlled. To achieve this control, the PM must be disrupted without unduly perturbing organelles (Schulz, 1990). The permeabilized cells are then bathed in medium that mimics cytosol, notably in its low [Ca2+]c (∼100 nM). Electroporation (Knight, 1981; Xie et al., 2013) and a variety of chemical means have been used to selectively permeabilize the PM. The chemicals achieve their PM-selectivity by interacting with cholesterol (e.g. saponin, digitonin, β-escin), which is enriched in the PM (Wassler et al., 1987), or as pore-forming toxins (e.g. α-toxin, streptolysin-O) that are too large to pass through the pores that they induce (Schulz, 1990).
After a protracted controversy (Babcock et al., 1979; Dehaye et al., 1980), analyses of permeabilized cells established that the ER, rather than mitochondria, is the major intracellular Ca2+ store in animal cells (Burgess et al., 1983). In an elegant study, saponin-permeabilized hepatocytes were bathed in cytosol-like medium with Ca2+ buffered to mimic the [Ca2+]c of an unstimulated cell. Each permeabilized cell was then shown to have the same Ca2+ content as an intact cell, and critically all of that Ca2+ was in the ER (Burgess et al., 1983). Hence, it is the ER from which most extracellular stimuli evoke Ca2+ release.
Analyses of insect salivary glands demonstrated that phosphoinositide turnover was required for extracellular stimuli to evoke Ca2+ signals (Berridge and Fain, 1979), and showed that IP3 was the first cytosolic product of receptor-stimulated phosphoinositide hydrolysis (Berridge, 1983). Hence, IP3 emerged as the likely messenger that links receptors in the PM to Ca2+ release from the ER (Fig. 1A). Permeabilized cells again were the subject of the decisive experiment: addition of IP3 to permeabilized pancreatic acinar cells stimulated release of Ca2+ from a non-mitochondrial Ca2+ store (Streb et al., 1983). It is now universally accepted that most IP3Rs reside in ER membranes, but IP3Rs can also mediate Ca2+ release from the Golgi apparatus (Aulestia et al., 2015; Pinton et al., 1998), the nuclear envelope (Foskett et al., 2007; Rahman et al., 2009; Stehno-Bittel et al., 1995) and perhaps from a nucleoplasmic reticulum (Echevarria et al., 2003). In some cells, a few IP3Rs (typically only 2–3 IP3Rs per cell) are also expressed in the PM, where they mediate Ca2+ entry (Dellis et al., 2006, 2008). In many studies, though not in all (Watras et al., 1991), the ER Ca2+ release evoked by IP3 was shown to be positively cooperative (e.g. Champeil et al., 1989; Marchant and Taylor, 1997; Meyer et al., 1988), suggesting a need for IP3 to bind to several IP3R subunits before the channel can open. A recent study using concatenated IP3R subunits showed that a defective IP3-binding site in only one of the four subunits prevents IP3R activation (Alzayady et al., 2016), leading to the conclusion that all four subunits of an IP3R must bind IP3 before the channel can open.
But IP3 binding is not alone sufficient to stimulate Ca2+ release through IP3Rs. Instead, IP3 binding primes IP3Rs to bind Ca2+, and Ca2+ binding then causes the channel to open (Adkins and Taylor, 1999; Marchant and Taylor, 1997) (Fig. 1B). Hence, IP3Rs require binding of two ligands, IP3 and Ca2+, to open. This dual regulation endows IP3Rs with their capacity to mediate regenerative Ca2+ signals through CICR. Again, it was analyses of permeabilized cells that provided the first evidence that Ca2+ release through IP3Rs is regulated by [Ca2+]c (Iino, 1987). High-resolution optical analyses of Ca2+ signals later revealed that within intact cells, IP3-evoked Ca2+ signals originate from elementary units that comprise a small cluster of IP3Rs (Smith and Parker, 2009; Thillaiappan et al., 2017). Opening of the first IP3R within a cluster is proposed to rapidly ignite the activity of some of its neighbours by CICR to generate a Ca2+ puff (Fig. 1C). As the stimulus intensity increases, Ca2+ spreading from one Ca2+ puff to another IP3R cluster can initiate further Ca2+ puffs, allowing the signal to spread across the cell as a regenerative Ca2+ wave (Marchant et al., 1999). The frequency of these global signals then increases with stimulus intensity (Thurley et al., 2014).
Structure-activity relationships (SAR), established by comparing the activities of a range of structurally-related chemical stimuli, are often used to probe the recognition properties of receptors. SAR analyses of the effects of IP3 analogues on Ca2+ release from permeabilized cells provided the first evidence that dephosphorylation of IP3 to (1,4)IP2 terminates IP3 activity (Burgess et al., 1984). The (1,3,4,5)IP4 that is produced when IP3 is phosphorylated by IP3 3-kinases was proposed to regulate IP3Rs (Loomis-Husselbee et al., 1996), but it is now clear that this phosphorylation also inactivates IP3 signalling through IP3Rs (Bird and Putney, 1996; Saleem et al., 2012). Hence, both endogenous pathways for IP3 metabolism effectively inactivate IP3 signalling to IP3Rs (Fig. 1A). SAR analyses of many analogues of IP3 and adenophostin A, a fungal metabolite that binds with high affinity to IP3Rs (Takahashi et al., 1994), established that a key feature of IP3R agonists is the presence of a vicinal 4,5-bisphosphate moiety (Fig. 1D) (Rossi et al., 2010, 2009; Saleem et al., 2012). All active inositol phosphate analogues have this 4,5-vicinal bisphosphate moiety (Fig. 1D).
There are no wholly selective antagonists of IP3Rs. Some ligands [heparin, 2-aminoethoxydiphenylborane (2-APB), xestospongin C and caffeine] have utility, but they all lack selectivity. Furthermore, heparin is not membrane-permeant, and results with xestospongin C are inconsistent (see Saleem et al., 2014). Addition of large substituents to the 2-O-position of IP3 produces partial agonists. Partial agonists are ligands that, once they have bound to IP3R, are less effective in causing the channel to open than full agonists like IP3 (Rossi et al., 2009). These SAR analyses of IP3 analogues modified at the 2-position, which again relied heavily on analyses of permeabilized cells, confirmed the importance of the extreme N-terminal region of the IP3R (the suppressor domain, SD; Fig. 1E) in IP3R activation (Rossi et al., 2009). They further suggest systematic strategies towards developing high-affinity antagonists of IP3Rs.
There is, therefore, a long history of experiments using permeabilized cells illuminating our understanding of IP3-evoked Ca2+ release. These studies first identified ER as the major intracellular Ca2+ store, they showed that IP3 evokes Ca2+ release from the ER, and that IP3Rs are regulated by Ca2+. Furthermore, they defined the biochemical steps that inactivate IP3 and, through SAR analyses, they have revealed ligands that have contributed to understanding the mechanisms of IP3R activation.
Analyses of IP3 binding to IP3Rs
Binding of IP3 to the four binding sites of the IP3R initiates the conformational changes that culminate in opening of the Ca2+-permeable pore (Alzayady et al., 2016; Chandrasekhar et al., 2016). These IP3 binding events are usually analysed by means of radioligand binding, which allows determination of binding affinities (as equilibrium dissociation constants, KD) for 3H-IP3 or any competing ligand, but a variety of other methods have also been successfully applied (Fig. 2, Box 2). KD values are important for comparison with functional analyses in revealing how ligands activate IP3Rs. Such analyses were, for example, critical in showing that the vicinal 4,5-bisphosphate of IP3 is essential for activity, whereas the 1-phosphate improves binding affinity (Fig. 1D) (Nahorski and Potter, 1989). Comparisons of SAR with binding analyses can also establish which bound ligands most effectively open the channel. Our comparisons of functional and 3H-IP3 equilibrium-competition binding analyses, for example, established that whereas IP3 is a full agonist that effectively gates the IP3R, other modified analogues of IP3 bind with high affinity to IP3R, but they are much less effective in causing the channel to open (Rossi et al., 2009). These partial agonists provide insight into the mechanisms of IP3R activation by demonstrating how large moieties at the 2-position of IP3 attenuate IP3R activation, (Rossi et al., 2009). They further suggest strategies for development of analogues that bind without activating IP3Rs (i.e. antagonists).
Analyses of IP3 binding allow affinities of IP3 or competing ligands to be determined (as equilibrium dissociation constants, KD, the concentration of IP3 at which 50% of binding sites are occupied) (Fig. 2). These analyses determine the relationship between the concentration of a ligand and the amount bound to IP3Rs. Radioligand binding, using 3H-IP3, is the most commonly used approach. Most methods used to determine specific binding of 3H-IP3 to IP3Rs require rapid separation of bound and free 3H-IP3, such that the equilibrium between free 3H-IP3, competing ligands and the IP3R is not perturbed by the separation procedure (filtration or centrifugation) (Fig. 2A,B). Measuring specific binding with different concentrations of 3H-IP3 allows the KD for 3H-IP3 to be determined, whereas measuring specific binding of 3H-IP3 in the presence of different concentrations of a competing ligand allow the KD of that ligand to be determined (Cheng and Prusoff, 1973). Advantages of these 3H-IP3 binding assays are their simplicity and applicability to IP3Rs within membranes, after detergent-solubilization or as IP3-binding fragments (Rossi et al., 2009). Scintillation proximity assays (SPA) avoid the need for separation steps because the SPA beads are impregnated with a scintillant, such that when IP3Rs are immobilized on the surface of the bead, only 3H-IP3 bound to an IP3R is detected (Fig. 2C) (Patel et al., 1996). More specialized methods allow analysis of ligand binding to IP3Rs without using radioligands. These methods include fluorescence polarization (FP), which uses a fluorescent analogue of IP3 to report the size of the molecule to which the fluorophore is attached. When free, the fluorescent IP3 is small and tumbles rapidly in solution, but when bound to a large IP3R fragment it tumbles more slowly. These changes can be detected using plane-polarized light (Fig. 2D) (Ding et al., 2010; Rossi and Taylor, 2013). Isothermal titration calorimetry (ITC), which measures heat exchange during IP3 binding, is another means of measuring ligand binding to IP3Rs without using 3H-IP3 (Fig. 2E) (de Azevedo and Dias, 2008). Limitations of both FP and ITC include the need for both specialised equipment and large amounts of purified protein.
Binding analyses also allow IP3R properties to be addressed under conditions where IP3-evoked Ca2+ release is not retained. This opportunity is particularly important during purification of IP3Rs for structural studies using either IP3R fragments for X-ray crystallography (Bosanac et al., 2002, 2005; Hamada et al., 2017; Lin et al., 2011; Seo et al., 2012) or, after detergent-solubilization of complete IP3Rs, for single-particle analysis by cryo-EM (Fan et al., 2015; Paknejad and Hite, 2018). In subsequent sections, we review progress towards understanding how IP3 binding leads to opening of the IP3R pore.
IP3 initiates IP3R activation by binding to the IP3-binding core
The route to determining IP3R structures began with the identification of specific, high-affinity, intracellular 32P-IP3-binding sites with recognition properties that matched those expected of the receptor through which IP3 evoked Ca2+ release (Baukal et al., 1985; Spät et al., 1986). Subsequent studies established that heparin competed with 3H-IP3 for these binding sites (heparin is a competitive antagonist of IP3), and that the sites were abundant in Purkinje cells of the cerebellum (Worley et al., 1987). Together, these observations allowed IP3Rs to be purified from cerebellum using heparin chromatography (Maeda et al., 1988; Supattapone et al., 1988). Functional reconstitution of the purified protein then established that it was alone sufficient to form an IP3-gated Ca2+ channel (Ferris et al., 1989; Maeda et al., 1991). Many additional proteins were later shown to associate with IP3Rs and modulate their responses to IP3 (Prole and Taylor, 2016). Screening of cDNA libraries from cerebellum then provided the complete primary sequence of IP3R1 (also known as ITPR1) (Furuichi et al., 1989; Mignery et al., 1989), and soon afterwards the other two IP3R subtypes, IP3R2 (ITPR2) (Südhof et al., 1991) and IP3R3 (ITPR3) (Blondel et al., 1993) were identified. Subsequent studies established that the three IP3R subunits (IP3R1–IP3R3) assemble to form homo-tetrameric and hetero-tetrameric channels (Monkawa et al., 1995), and confirmed that the core properties of all IP3R subtypes are similar: each forms a large-conductance Ca2+-permeable channel that is gated by binding of IP3 and Ca2+ (Foskett, 2010), and each generates Ca2+ puffs (Mataragka and Taylor, 2018). The subtypes are, however, differentially expressed, and they differ in their affinities for IP3 (Iwai et al., 2007), sensitivity to Ca2+ regulation (Foskett, 2010) and in whether they are modulated by additional regulators (Prole and Taylor, 2016). Furthermore, the functional consequences of mutant or defective IP3Rs differ among subtypes (see Terry et al., 2018). IP3R1 has so far been the major focus of the structural studies.
Deletion analyses (Mignery and Südhof, 1990) and expression of IP3R fragments in bacteria (Yoshikawa et al., 1996) established that each IP3R subunit has a single IP3-binding site, the IBC, formed by residues 224–604 towards the N-terminal of the primary sequence (with a total of ∼2750 residues) (Fig. 1E). The identification of four IP3-binding sites in each IP3R tetramer, and the demonstration that all four are required for IP3 to evoke Ca2+ release (Alzayady et al., 2016), provided an explanation for the widely observed cooperative responses to IP3 (Champeil et al., 1989; Meyer et al., 1988; Parker and Miledi, 1989). Subsequent studies identified residues within the IBC that are required for IP3 binding, notably the residues that bind to the critical 4- and 5-phosphate groups of IP3 (Furutama et al., 1996). These residues are conserved in IP3Rs, but not in RyRs (Bosanac et al., 2002; Seo et al., 2012). It was also shown that the SD inhibits IP3 binding (Uchida et al., 2003), which aligns with the importance of the SD in coupling IP3 binding to channel gating (Rossi et al., 2009): IP3Rs without an SD bind IP3 with high affinity, but they do not release Ca2+ (Uchida et al., 2003).
Examination of high-resolution crystal structures of N-terminal fragments of the IP3R directly revealed both the determinants of IP3 binding and the initial steps in IP3R activation. The two domains (α and β) of the IBC form a clam-shaped structure, within which conserved residues bind to IP3 (Bosanac et al., 2002). The 1- and 5-phosphates of IP3 interact predominantly with residues in IBC-α, whereas the 4-phosphate interacts with IBC-β (Fig. 1D,E). Interaction of the critical 4- and 5-phosphates with opposing sides of the ‘clam’ allows IP3 to partially close the clam and initiate IP3R activation (Hamada et al., 2017; Lin et al., 2011; Paknejad and Hite, 2018; Seo et al., 2012). This interpretation, which elegantly reveals the structural basis of the SAR, is supported by results with an adenophostin A analogue in which an alternative contact with the α-domain substitutes for loss of the usual phosphate (Sureshan et al., 2012).
In the isolated N-terminal domain, the SD is firmly anchored to IBC-α by an extensive interface and more loosely associated with IBC-β (Fig. 1E). Hence, when IP3 causes the IBC clam to close, the SD moves with IBC-α, which was predicted to disrupt interaction of an exposed SD loop, the ‘hot spot’ loop (Yamazaki et al., 2010) with IBC-β of a neighbouring subunit (Seo et al., 2012). In RyR, too, these inter-subunit interactions between N-terminal domains are weakened during receptor activation (des Georges et al., 2016). The resulting weakening of interactions between subunits may contribute to channel gating. This is supported by evidence that Ca2+-binding protein 1 (CABP1), which inhibits IP3R gating, rigidifies these interactions between IP3R subunits (Li et al., 2013). However, within the constraints of a full-length IP3R, strong inter-subunit interactions between the SD and IBC-β might constrain the SD, such that IBC-α moves when IP3 closes the clam (Paknejad and Hite, 2018). Identification of the sites to which IP3 binds, which relied heavily on radioligand binding analyses, set the scene for the structural analyses that allow researchers to seek to understand how IP3 binding opens the pore of the IP3R. We consider recent progress with such structural analyses in the next section.
Structures of complete IP3 and ryanodine receptors
Single-particle analysis of cryo-EM images has allowed determination of the structures of the complete IP3R1 in a closed state (Fan et al., 2015), of IP3R3 with and without IP3 and Ca2+ bound (Paknejad and Hite, 2018), and of RyRs in different states (des Georges et al., 2016; Efremov et al., 2015; Peng et al., 2016; Yan et al., 2015; Zalk et al., 2015), and has begun to reveal the workings of the pore regions of these related channels. The results also tentatively suggest how IP3 binding might lead to opening of the IP3R pore.
The IP3R has a structure reminiscent of a square mushroom. Much of the stalk is embedded in the ER membrane and the cap, with a diameter of ∼25 nm, extends at least 13 nm into the cytosol (Fan et al., 2015). The large size is significant because it might exclude IP3Rs from the narrow junctions between ER and the PM (Thillaiappan et al., 2017), whereas at other junctions, between ER and mitochondria for example (Csordás et al., 2018), it places the head of the IP3R, from which Ca2+ exits, very close to the neighbouring organelle.
The cytosolic entrance to the central cavity of the IP3R is surrounded by the N-terminal domains (SD and IBC-β, Fig. 3). IBC-α forms part of a larger domain (ARM1) that curves to the edge of the cap and interacts with two large curved domains (ARM2 and ARM3) that comprise most of the remaining cytosolic structure and form the underside of the mushroom cap (Fig. 3). Within the ER membrane, there are 24 IP3R transmembrane domains (TMDs), six from each subunit (Fan et al., 2015). However, recent structural analyses of both IP3R (Paknejad and Hite, 2018) and RyR1 (des Georges et al., 2016) identified a pair of additional helices (between TMD1 and TMD2 of IP3R3) that challenge the accepted view that there are six TMDs per subunit. The TMD region, similar in structure to voltage-gated ion channels, is very similar (though not identical) (Baker et al., 2017) in RyRs and IP3Rs. The ion-conducting path is lined by the four tilted TMD6 helices and a short (∼1 nm) ‘selectivity filter’ at the luminal end through which hydrated cations must pass in single file. The selectivity filter, its supporting pore-loop helix and a flexible luminal loop are all formed by residues linking TMD5 to TMD6. Near the cytosolic end of TMD6, a narrow hydrophobic constriction blocks the movement of ions in the closed channel (Fan et al., 2015) (Fig. 3). The hydrophobic side chains of these residues must move for the pore to open. Opening of the RyR pore is associated with splaying and bowing of TMD6, such that the hydrophobic side-chain of a residue that occludes the cytosolic end of the closed pore is displaced, opening a hydrophilic path that allows passage of a hydrated Ca2+ ion. Similar mechanisms may be associated with opening of the IP3R pore.
TMD6 is supported by TMD5, which in turn is buttressed by the TMD1–TMD4 bundle of the adjacent subunit. The short cytosolic TMD4–TMD5 helical linker aligns along the ER membrane behind the TMD6 helices, holding them in place. In the closed RyR1 channel, this linker tightly encircles the cytosolic end of the TMD6 bundle, restricting its movement, but this grip is relaxed as the channel opens, freeing TMD6 to move and allowing the pore to dilate (des Georges et al., 2016). In both IP3R and RyR, TMD6 extends well beyond the ER membrane (∼1.5 nm in IP3R) and then terminates in a pair of short α-helices (the linker domain, LNK, in IP3R) that includes a Zn2+-finger motif that aligns parallel with the ER membrane (des Georges et al., 2016; Fan et al., 2015; Paknejad and Hite, 2018). In IP3R, but notably not in RyR, the entwined TMD6 helices then continue beyond the LNK domain to the cap of the mushroom, where each contacts the IBC-β domain of a neighbouring subunit (Fan et al., 2015). Hence, structures formed by the TMD5–TMD6 loop guard the luminal entrance to the pore, whereas the cytosolic vestibule is formed by the extended TMD6. Each of these regions is enriched in acidic residues that probably contribute to the cation selectivity of IP3R and RyR (des Georges et al., 2016; Fan et al., 2015; Paknejad and Hite, 2018).
A conserved Ca2+-binding site is present in both RyR (des Georges et al., 2016) and IP3R (Paknejad and Hite, 2018). The site is formed, in the case of IP3R, by residues near the C-terminal end of ARM3 and by another residue contributed by the LNK domain (Fig. 3). In RyR, the equivalent residues are proposed to coordinate the Ca2+ required for stimulation (des Georges et al., 2016). The same may hold true for IP3Rs, but this has yet to be tested. A conserved glutamate residue on the bottom surface of the ARM3 domain (Glu2101 in IP3R1) previously suggested to mediate Ca2+ regulation of IP3R (Miyakawa et al., 2001) and RyR (Fessenden et al., 2001), does not contribute to Ca2+ binding to this site, but it does stabilize the interaction between the cooperating domains in RyR1 (des Georges et al., 2016). A second Ca2+-binding site was identified in the structure of IP3R3, and again it is formed by residues that are contributed by different domains (ARM3 and the α-helical domain linking ARM1 to ARM2) (Paknejad and Hite, 2018) (Fig. 3B). Formation of both Ca2+-binding sites requires movement of the contributing domains from their positions in the apo-state (i.e. IP3R without bound IP3), so as to bring the Ca2+-coordinating residues into register (Paknejad and Hite, 2018). This important observation is consistent with evidence that IP3 controls IP3R gating by regulating Ca2+ binding (Fig. 1B).
Taken together, analyses of the structures of full-length IP3Rs have allowed researchers to define where IP3 binds, identify Ca2+-binding sites that may mediate Ca2+ regulation, and establish that hydrophobic residues projecting into the pore must move to allow Ca2+ to pass.
Towards understanding how IP3 and Ca2+ binding open the IP3R pore
The only contacts between the large cytosolic structures of the IP3R and its channel region are the C-terminal end of ARM3 and the LNK domain (Fig. 3) (Fan et al., 2015). There are similar contacts in RyR (des Georges et al., 2016). In both IP3R and RyR, this critical nexus comprises a platform of interleaved structures: the C-terminus of the ARM3 domain (the intervening lateral domain, ILD) forms a ‘thumb-and-fingers’ arrangement of an upper thumb abutting the bulk of ARM3, and an α-helical pair of fingers lying below and forming a cavity into which the LNK domain inserts (Fig. 3) (Fan et al., 2015). Mutations within the thumb disrupt IP3R function (Hamada et al., 2017). The LNK domain also wraps around the thumb and contributes a residue to the Ca2+-binding site at the base of the ARM3 domain.
How, then, does IP3 binding to the IBC cause hydrophobic pore residues some 7 nm distant to move and allow Ca2+ to pass from the ER lumen to the cytosol (Fan et al., 2015)? Recalling that IP3 primes IP3Rs to bind Ca2+, which then triggers channel opening (Adkins and Taylor, 1999) (Fig. 1B), it seems reasonable to speculate that IP3 binding to the IBC is communicated to the Ca2+-binding site at the ILD–LNK nexus and thence to the pore (Paknejad and Hite, 2018). IP3 binding closes the clam-like IBC, and, with IBC-β held firmly in place by inter-subunit interactions at the top of the mushroom, IBC-α moves and initiates conformational changes throughout the associated ARM domains. These changes include disruption of inter-subunit interaction between ARM1 and ARM2 domains, and rotation of the LNK domains (Paknejad and Hite, 2018). Here, the need for the SD is attributed to its role in stabilizing inter-subunit interactions to provide a fixed structure against which movement of IBC-α can leverage conformational changes through the ARM domains (Paknejad and Hite, 2018). Given the essential role of the SD in IP3R activation, an alternative possibility was that the direct contact between the SD and ARM3 might mediate communication between N-terminal regions and the ILD. However, the SD–ARM3 interaction occurs through the handle of the hammer-like SD, which can be deleted without impairing IP3R function (Yamazaki et al., 2010). Another possibility was that interaction between IBC-β and the C-terminal α-helical domain (CTD), which is unique to IP3R, might communicate IP3 binding to the LNK domain. However, this scheme is difficult to reconcile with functional IP3R/RyR chimeras (Seo et al., 2012) since the RyR structure does not have an extended CTD, and with evidence that deletion of residues within the CTD that interact with IBC-β do not prevent IP3-evoked Ca2+ release (Hamada et al., 2017; Schug and Joseph, 2006). Whatever the exact path from IBC to the ILD–LNK nexus is, IP3-evoked conformational changes appear to reconfigure the Ca2+-binding site formed at the LNK–ARM3 interface to allow Ca2+ binding (Paknejad and Hite, 2018), thereby providing a plausible mechanism for IP3 priming IP3Rs to respond to Ca2+ (Fig. 1B).
We conclude that analyses of IP3 binding contributed to defining the SAR for IP3Rs and to quantitative comparisons of the relationship between binding and channel activation, but most significantly they allowed IP3Rs to be identified during their purification, which paved the way to cloning and molecular manipulation of IP3Rs, and to structural studies. The latter have allowed investigators to establish that IP3Rs are huge tetrameric structures, wherein IP3 binding closes a clam-like IBC. This conformational change is communicated to a critical nexus between interleaved structures from the cytosolic and channel domains. IP3 binding probably stabilizes Ca2+ binding to this nexus, leading to rearrangement of the pore, such that occluding hydrophobic residues are displaced to allow the passage of Ca2+ from the ER lumen to the cytosol.
Lessons from planar lipid bilayers and patch-clamp recording
Electrical recordings from ion channels, most often by means of patch-clamp recording (Box 3) (Lape et al., 2008; Neher, 1992), allow the opening and closing of single channels to be recorded with sub-millisecond resolution, and they allow their ion permeation properties to be defined. Because the intracellular location of RyR and IP3R in the ER presents a formidable barrier to such recordings (Jonas et al., 1997), two alternative approaches have been used.
Patch-clamp recording allows the opening and closing of single ion channels to be recorded with exquisite sensitivity (Neher, 1992). Usually these recordings are made at the plasma membrane (PM), but that is not applicable to single-channel recordings from IP3Rs, most of which are expressed in the ER. However, the outer nuclear membrane (ONM) is continuous with the ER membrane, and IP3Rs are expressed in the ONM. A glass microelectrode applied to the ONM of an isolated nucleus allows single-channel recording from IP3Rs trapped within. By excising the patch from the intact nucleus, it is possible to make recordings with the IP3-binding site of the IP3R exposed to either the interior of the patch-pipette patch or (with greater difficulty) to the bath solution (Mak et al., 2007). The latter allows rapid application of IP3 or Ca2+ to the cytosolic surface. K+ or Cs+ are commonly used as charge-carriers for patch-clamp recording because they provide large currents and, unlike Ca2+, they do not regulate IP3R gating. These patch-clamp methods allow the ion selectivity and conductance of IP3Rs to be determined. By examining the sequence of channel openings and closing, gating schemes can be developed that seek to explain how regulators of IP3Rs (like IP3 and Ca2+) move the channel through different closed states to its open state (Mak and Foskett, 2014; Rahman et al., 2009). Image reproduced, with permission, from Rossi et al. (2012).
Planar lipid bilayers
The first approach, which involves reconstitution of ER vesicles or solubilized IP3Rs into planar lipid bilayers, provided the first measurements of currents through IP3R (Bezprozvanny et al., 1994, 1991; Bezprozvanny and Ehrlich, 1994; Ehrlich and Watras, 1988; Maeda et al., 1991). These analyses established that IP3R, like RyR, are large-conductance cation channels with relatively low selectivity for Ca2+. Both features are important in allowing IP3R to generate large local cytosolic Ca2+ signals: the large conductance allows an open IP3R to pass ∼500,000 Ca2+ ions per second (Foskett et al., 2007), whereas the weak selectivity might allow a counter-flux of K+ to dissipate the electrical gradient that is formed as Ca2+ leaves the ER and which would otherwise rapidly terminate Ca2+ release (Zsolnay et al., 2018). The short, wide selectivity filter and large vestibules with abundant acidic residues probably provide the structural basis of these ion permeation properties (Fan et al., 2015). Bilayer analyses also allowed confirmation of the biphasic regulation of IP3R1 by cytosolic Ca2+ (Bezprozvanny et al., 1991). A potential problem with recordings from planar lipid bilayers is that solubilization and/or reconstitution could lead to loss of accessory proteins or perturbation of structure. Maximal open probabilities recorded from IP3Rs in bilayers, for example, are much lower than in patch-clamp recordings, and bilayer recordings of IP3R2 and IP3R3 failed to capture the inhibitory effect of cytosolic Ca2+ (Hagar et al., 1998; Ramos-Franco et al., 2000).
The second approach to obtaining electrical recordings from IP3R exploits the fact that the ER is continuous with the outer nuclear membrane (ONM) (Box 3) (Dingwall and Laskey, 1992). Hence, patch-clamp recording from the ONM allows analysis of IP3R in a native membrane, albeit not the ER (Mak et al., 2013; Rahman and Taylor, 2010) (Box 3). Examination of these recordings, which have been applied to both native and heterologously expressed IP3Rs (Betzenhauser et al., 2008; Cheung et al., 2010; Foskett et al., 2007; Marchenko et al., 2005; Rahman et al., 2009), confirmed the ion permeation properties of IP3Rs and the biphasic regulation of all IP3R subtypes by Ca2+. The recordings have also suggested complex gating schemes wherein IP3 drives bursts of IP3R activity by extending the duration of sequences of openings and shortening the gaps between the bursts (Gin et al., 2009; Ionescu et al., 2007).
Another application of nuclear patch-clamp recording is provided by our work, where we showed that IP3Rs within patches that fortuitously contained several IP3Rs behave differently to patches with only a single IP3R (Rahman et al., 2009). This led to our proposal that low concentrations of IP3, perhaps arising from occupancy of only some of the four IP3-binding sites, trigger IP3R clustering (Rahman et al., 2009). The clustered IP3Rs, we suggest, are better placed than lone IP3Rs to benefit from CICR when a near neighbour opens to release Ca2+ and so provide the second stimulus that is needed for IP3R opening (Fig. 1B). Effects of clustering on the IP3 and Ca2+ sensitivity of IP3Rs reinforce the propensity of clustered IP3Rs to amplify Ca2+ signals by CICR. These proposals have been challenged (Rahman et al., 2011; Smith et al., 2009; Vais et al., 2011) and our own recent work suggests that even in unstimulated cells there are pre-existing clusters of IP3Rs, each typically comprising about eight IP3Rs (Thillaiappan et al., 2017). Our revised proposal therefore envisages that IP3Rs are, as we have shown, loosely clustered in unstimulated cells (Thillaiappan et al., 2017) and that IP3 might then cause IP3Rs within the cluster to huddle more closely and so be more likely to respond to Ca2+ released by a neighbour.
Throughout the long history of analyses of intracellular Ca2+ signalling, there has been a productive interplay between studies of intact tissues and of biological systems extracted from intact cells (ex cellula). These approaches have revealed that the ER is the major intracellular Ca2+ store and allowed identification of the enormous channels (RyR and IP3R) that mediate Ca2+ release from the ER. We are now fast approaching an understanding of how IP3 binding leads, through its interactions with Ca2+ binding, to opening of the IP3R. In parallel with these approaches, developments in optical microscopy have provided opportunities to examine IP3-evoked Ca2+ release with exquisite temporal and spatial resolution in intact cells. We can surely look forward to these analyses converging with structural analyses in situ to provide a comprehensive understanding of IP3Rs in living cells.
This work was supported by the Wellcome Trust (grant number 101844) and the Biotechnology and Biological Sciences Research Council (grant number BB/P005330/1).
The authors declare no competing or financial interests.