© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs222463. doi:10.1242/jcs.222463 REVIEW SPECIAL ISSUE: RECONSTITUTING CELL BIOLOGY IP3 receptors – lessons from analyses ex cellula Ana M. Rossi and Colin W. Taylor* ABSTRACT and Ca2+ held within intracellular stores are entangled. For cardiac Inositol 1,4,5-trisphosphate receptors (IP Rs) are widely expressed muscle, depolarization of the plasma membrane (PM) causes 3 2+ intracellular channels that release Ca2+ from the endoplasmic voltage-gated Ca channels (Cav1.2, also known as CACNA1C) to 2+ reticulum (ER). We review how studies of IP Rs removed from their open, and the local increase in cytosolic free Ca concentration 3 2+ 2+ 2+ intracellular environment (‘ex cellula’), alongside similar analyses of ([Ca ]c) is then amplified by Ca -induced Ca release (CICR) ryanodine receptors, have contributed to understanding IP R through type 2 ryanodine receptors (RyR2) in the sarcoplasmic 3 2+ behaviour. Analyses of permeabilized cells have demonstrated that reticulum (Bers, 2002) (Fig. 1A). CICR and the local Ca 2+ signalling that is required to avoid CICR from becoming the ER is the major intracellular Ca store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the explosive have become recurrent themes in the field of Ca2+ signalling (Rios, 2018). Fluorescent Ca2+ indicators and 4,5-phosphates of IP3 are essential for activating IP3Rs, and optical microscopy now allow Ca2+ sparks, local Ca2+ signals facilitated IP3R purification and cloning, which paved the way for evoked by a small cluster of RyRs, to be measured with exquisite structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established subcellular resolution in cardiac muscles (Cheng and Lederer, 2+ 2008). However, it was studies of permeabilized cells (‘skinned’ that IP3Rs have a large conductance and select weakly between Ca fibres) that provided the first evidence for CICR in muscle (Endo and other cations. Structural analyses are now revealing how IP3 et al., 1970; Fabiato and Fabiato, 1979). Analyses of RyRs that were binding to the N-terminus of the tetrameric IP3R opens the pore reconstituted into planar lipid bilayers first showed that RyRs form ∼7 nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved large-conductance cation channels that are biphasically regulated by 2+ domains contributed by structures associated with the pore and cytosolic Ca (Lai et al., 1988; Meissner, 2017). Finally, analyses cytosolic domains, which together contribute to a Ca2+-binding site. of RyR fragments by X-ray crystallography (Van Petegem, 2014) These structural analyses provide evidence to support the suggestion and of complete RyRs by cryo-electron microscopy (des Georges 2+ et al., 2016; Efremov et al., 2015; Peng et al., 2016; Yan et al., 2015; that IP3 gates IP3Rs by first stimulating Ca binding, which leads to pore opening and Ca2+ release. Zalk et al., 2015) are revealing the structural basis of RyR behaviour. KEY WORDS: Bilayer recording, Ca2+ channel, Endoplasmic Progress towards understanding the second major family of 2+ reticulum, Ion channel structure, IP3 receptor, Nuclear patch-clamp, intracellular Ca -release channels, the IP3Rs, began with an Permeabilized cell, Radioligand binding, Ryanodine receptor influential review in which a causal link between receptor- stimulated turnover of phosphatidylinositol and Ca2+ signalling Introduction was proposed (Michell, 1975). Subsequent work established Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine that many receptors stimulate phospholipases C, which cleave 2+ receptors (RyR) are the two major families of intracellular Ca - phosphatidylinositol 4,5-bisphosphate to produce IP3 and release channels in animal cells (Fig. 1A). IP3Rs are expressed in diacylglycerol (Berridge, 1993) (Fig. 1A). IP3 provides the link to most cells, whereas RyRs have a more restricted distribution. RyRs Ca2+ signalling; not, as first envisaged, by directly stimulating Ca2+ are most abundant in excitable cells, notably in striated muscle, entry across the PM (Michell, 1975), but by stimulating Ca2+ release where they contribute to excitation–contraction coupling (Fig. 1A) from the endoplasmic reticulum (ER) through IP3Rs (Berridge (Van Petegem, 2014). In this Review, we focus on IP3Rs, and and Irvine, 1984; Streb et al., 1983). Another influential review 2+ 2+ how methods applied to IP3Rs removed from intact cells have suggested the link between IP3-evoked Ca release and Ca entry 2+ contributed to our understanding of IP3R behaviour. Progress in our across the PM, and proposed that loss of Ca from the ER 2+ understanding of IP3Rs and RyRs has advanced in parallel, and with stimulated Ca entry (Putney, 1986). The workings of this store- this progress it became clear that the two families share structural operated Ca2+ entry (SOCE) pathway are now clear: dissociation of and functional features (Baker et al., 2017; Seo et al., 2012). Hence, Ca2+ from the luminal EF-hand motif of a protein embedded in the despite our focus on IP3Rs, we draw also on evidence from analyses ER membrane, stromal interaction molecule 1 (STIM1), causes of RyRs. STIM1 to oligomerize and expose a cytosolic domain, through Classic work by Sydney Ringer demonstrated that cardiac muscle which it stimulates opening of a Ca2+-selective channel in the PM contraction requires extracellular Ca2+ (Ringer, 1883). This was, (Feske et al., 2006; Prakriya and Lewis, 2015). The Ca2+ channel with the benefit of hindsight, the first of many studies to show that that mediates SOCE is a hexameric assembly of Orai subunits (Hou the contributions to physiological responses of extracellular Ca2+ et al., 2012; Yen and Lewis, 2018), grandiloquently named from Greek mythology after the keepers of heaven (Feske et al., 2006). 2+ Department of Pharmacology, University of Cambridge, Tennis Court Road, IP3Rs and RyRs are biphasically regulated by cytosolic Ca Cambridge CB2 1PD, UK. (Bezprozvanny et al., 1991). For IP3Rs exposed to IP3, a modest 2+ 2+ increase in [Ca ]c stimulates opening, whereas a higher [Ca ]c is *Author for correspondence ([email protected]) inhibitory (Foskett et al., 2007; Iino, 1990). Hence IP3Rs, at least C.W.T., 0000-0001-7771-1044 once they have bound IP3 (Alzayady et al., 2016), can, like RyRs, Journal of Cell Science 1 REVIEW Journal of Cell Science (2019) 132, jcs222463. doi:10.1242/jcs.222463 A GPCRRTK Cav1.1 Cav1.2 PM I II III IV I II III IV β γ PLC PLC G protein Conformational coupling CICR IP2 IP4 IP3 Deactivation RyR1 RyR2 IP3 R Cytosol ER/ SR ER/SR lumen B IP Ca2+ D Essential 3 IP 3 P Cytosol P Enhances P affinity ER lumen E IP3R C CICR SD IBC TMDs β α 1 223 604 Cytosol Loop ER lumen SD Ca2+ puff IBC-β 5 4 IBC-α IP3 2+ Fig. 1. Ca release by IP3 and ryanodine receptors. (A) Many receptors in the plasma membrane (PM), including G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), stimulate phospholipases C (PLC), causing hydrolysis of the PM lipid, phosphatidylinositol 4,5-bisphosphate, into diacylglycerol and IP3.IP3 binds to each of the four IP3-binding sites of the tetrameric IP3R to initiate conformational changes that lead to channel opening and 2+ release of Ca from the ER. IP3 is deactivated by phosphorylation to IP4 or dephosphorylation to IP2. RyRs are close relatives of IP3Rs, but they are predominantly expressed in the sarcoplasmic reticulum of skeletal (RyR1) and cardiac (RyR2) muscle. Each RyR is activated when depolarization of thePM 2+ 2+ activates voltage-gated Ca channels (Cav1). RyR1 are directly activated by conformational coupling to CaV1.1 (CACNA1S), whereas Ca entering cardiac 2+ 2+ myocytes through Cav1.2 activates RyR2 through Ca -induced Ca release (CICR). Structures from Electron Microscopy Data Bank: IP3R, EMD-5278 (Ludtke 2+ et al., 2011), RyR1, EMD-1275 (Ludtke et al., 2005). (B) IP3 binding is not alone sufficient to activate IP3Rs. IP3 binding primes IP3Rs to bind Ca and that 2+ leads to channel opening. All four IP3-binding sites must be occupied for the pore to open, but it is not yet known how many Ca -binding sites must be occupied 2+ 2+ (we show four for simplicity). (C) Dual regulation of IP3Rs by IP3 and Ca allows IP3Rs to propagate regenerative Ca signals by CICR. Local CICR activity 2+ within a small cluster of IP3Rs generates a Ca puff. (D) The vicinal 4,5-bisphosphate moiety of IP3 is essential for activity, whereas the 1-phosphate enhances affinity. (E) IP3 is recognised by the IP3-binding core (IBC) of IP3R. The essential 4- and 5-phosphates of IP3 interact with opposing sides of the clam-like IBC to cause clam closure. The loop of the suppressor domain (SD) interacts with IBC-β of a neighbouring subunit (Seo et al., 2012). A–C modified from Taylor et al. (2014), and E reproduced, with permission, from Seo et al. (2012). 2+ mediate CICR (Fig. 1B,C). As with RyRs, IP3Rs assemble into Ca -release channels and the focus of this Review, they are not the 2+ clusters, within which opening of one IP3R ignites the activity of its only intracellular Ca channels. Brief descriptions of additional neighbours to generate local ‘Ca2+ puffs’ (Fig. 1C) (Smith and intracellular Ca2+ channels are provided in Box 1. Parker, 2009; Thillaiappan et al., 2017), analogous to Ca2+ sparks in The productive interplay between studies of minimally perturbed muscle. These behaviours illustrate some of the many similarities tissue, facilitated by a plethora of Ca2+ indicators (Lock et al., 2015), between IP3Rs and RyRs, which include their close structural fluorescent proteins (Rodriguez et al., 2017) and fluorescence relationship (Baker et al., 2017; Seo et al., 2012; Van Petegem, microscopy techniques (Thorn, 2016), alongside analyses of 2+ 2014).