Volume 171, Issue 13 p. 3298-3312
RESEARCH PAPER
Open Access

Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor

Huma Saleem

Huma Saleem

Department of Pharmacology, University of Cambridge, Cambridge, UK

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Stephen C Tovey

Stephen C Tovey

Department of Pharmacology, University of Cambridge, Cambridge, UK

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Tadeusz F Molinski

Tadeusz F Molinski

Department of Chemistry, University of California, Davis, CA, USA

This article was amended (10 July 2014) after publication to correct the forename of this author.Search for more papers by this author
Colin W Taylor

Corresponding Author

Colin W Taylor

Department of Pharmacology, University of Cambridge, Cambridge, UK

Correspondence

Colin W Taylor, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K. E-mail: [email protected]

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First published: 13 March 2014
Citations: 83

Abstract

Background and Purpose

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Interactions of the commonly used antagonists of IP3Rs with IP3R subtypes are poorly understood.

Experimental Approach

IP3-evoked Ca2+ release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP3R was measured using a luminal Ca2+ indicator. The effects of commonly used antagonists on IP3-evoked Ca2+ release and 3H-IP3 binding were characterized.

Key Results

Functional analyses showed that heparin was a competitive antagonist of all IP3R subtypes with different affinities for each (IP3R3 > IP3R1 ≥ IP3R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP3R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP3R1 without affecting IP3 binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP3-evoked Ca2+ release via any IP3R subtype.

Conclusions and Implications

Heparin competes with IP3, but its access to the IP3-binding core is substantially hindered by additional IP3R residues. These interactions may contribute to its modest selectivity for IP3R3. Practicable concentrations of caffeine and 2-APB inhibit only IP3R1. Xestospongins do not appear to be effective antagonists of IP3Rs.

Abbreviations

  • 2-APB
  • 2-aminoethoxydiphenyl borate
  • AdA
  • adenophostin A
  • CLM
  • cytosol-like medium
  • CPA
  • cyclopiazonic acid
  • ER
  • endoplasmic reticulum
  • HBS
  • HEPES-buffered saline
  • IBC
  • IP3-binding core (residues 224-604)
  • IP3
  • inositol 1,4,5-trisphosphate
  • IPTG
  • isobutyl-β-D-thiogalactoside
  • NT1-3
  • residues 1-604 of IP3R1-3
  • SERCA
  • sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
  • TEM
  • Tris-EDTA medium
  • Introduction

    Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels expressed in the membranes of the endoplasmic reticulum (ER) in most eukaryotic cells (Berridge, 1993; Taylor et al., 1999; Foskett et al., 2007; nomenclature follows Alexander et al., 2013). IP3Rs are essential links between the many extracellular signals that stimulate PLC and initiation of cytosolic Ca2+ signals triggered by IP3-evoked Ca2+ release from the ER. Three genes encode closely related IP3R subunits in vertebrates, whereas invertebrates have only a single IP3R gene (Taylor et al., 1999). Each of the three vertebrate IP3R subtypes encodes a large polypeptide of about 2700 residues, and they share about 70% amino acid sequence identity (Foskett et al., 2007). Within each IP3R subunit, IP3 binds to a clam-like IP3-binding core (IBC; residues 224–604 in IP3R1) (Bosanac et al., 2002) near the N-terminus. IP3 binding to the IBC re-orients its relationship with the associated suppressor domain (residues 1–223). That rearrangement disrupts interactions between adjacent subunits within the tetrameric IP3R leading to gating of the Ca2+-permeable channel (Seo et al., 2012). This central channel of each tetrameric IP3R is formed by transmembrane helices and their associated re-entrant loops. These pore-forming structures lie towards the C-terminal of each subunit. How IP3-evoked re-arrangement of N-terminal domains of the IP3R leads to opening of the pore is not yet resolved, although it is likely to be conserved in all IP3R subtypes and broadly similar for the other major family of intracellular Ca2+ channels, ryanodine receptors (Seo et al., 2012).

    Most cells express mixtures of IP3R subtypes, although tissues differ in which complements of IP3R subunits they express (Taylor et al., 1999). Furthermore, the subunits assemble into both homo-tetrameric and hetero-tetrameric structures (Wojcikiewicz and He, 1995). Although all IP3Rs are built to a common plan and they are all regulated by IP3 and Ca2+ (Foskett et al., 2007; Seo et al., 2012), the subtypes are subject to different modulatory influences (Patterson et al., 2004; Higo et al., 2005; Foskett et al., 2007; Betzenhauser et al., 2008; Wagner and Yule, 2012) and they are likely to fulfil different physiological roles (Matsumoto et al., 1996; Hattori et al., 2004; Futatsugi et al., 2005; Tovey et al., 2008; Wei et al., 2009). It is, however, difficult to disentangle the physiological roles of IP3R subtypes in cells that typically express complex mixtures of homo- and hetero-tetrameric IP3Rs. There are no ligands of IP3Rs that usefully distinguish among IP3R subtypes (Saleem et al., 2012; 2013) and nor are there effective antagonists that lack serious side effects (Michelangeli et al., 1995). Heparin (Ghosh et al., 1988), caffeine (Parker and Ivorra, 1991), 2-aminoethoxydiphenyl borate (2-APB) (Maruyama et al., 1997) and Xestospongins (Gafni et al., 1997) have all been widely used to inhibit IP3-evoked Ca2+ release, but each has its limitations (see Results). Furthermore, the interactions of these antagonists with IP3R subtypes have not been assessed. Peptides derived from myosin light-chain kinase (Nadif Kasri et al., 2006; Sun and Taylor, 2008), the N-terminal of IP3R1 (Sun et al., 2013) or the BH4 domain of bcl-2 (Monaco et al., 2012) also inhibit IP3-evoked Ca2+ release. These peptides are unlikely to provide routes to useful IP3R antagonists because they are effective only at high concentrations and they need to be made membrane-permeable. A naturally occurring protein that inhibits IP3 binding to IP3R, IRBIT (Ando et al., 2003), has the same limitations as an experimental tool, and it is effective only when phosphorylated. Many other drugs inhibit IP3-evoked Ca2+ release, but none of these has found widespread use (see Michelangeli et al., 1995; Bultynck et al., 2003).

    In the present study, we provide the first systematic analysis of the interactions between IP3R subtypes and each of the commonly used antagonists. We use DT40 cell lines stably expressing only a single mammalian IP3R subtype to define the effects of these antagonists on IP3-evoked Ca2+ release via each IP3R subtype.

    Methods

    Measurement of IP3-evoked Ca2+ release

    We used DT40 cells lacking endogenous IP3Rs (Sugawara et al., 1997), but stably expressing rat IP3R1 (GenBank accession number GQ233032.1; Pantazaka and Taylor, 2011), mouse IP3R2 (GU980658.1; Tovey et al., 2010) or rat IP3R3 (GQ233031.1; Rahman et al., 2009). Cells were grown in suspension in RPMI 1640 medium supplemented with 10% FBS, 1% heat-inactivated chicken serum, 2 mM glutamine and 50 μM 2-mercaptoethanol at 37°C in humidified air containing 5% CO2. Cells were used or passaged when they reached a density of ∼1.5 × 106 cells mL−1.

    A low-affinity Ca2+ indicator trapped within the ER of permeabilized DT40 cells was used to measure IP3-evoked Ca2+ release (Tovey et al., 2006; Saleem et al., 2012). Briefly, the ER was loaded with indicator by incubating cells (∼5 × 107 mL−1) in the dark with Mag-fluo-4AM (20 μM) in HEPES-buffered saline (HBS) containing 0.02% (v/v) Pluronic F127 for 1 h at 22°C. HBS had the following composition: 135 mM NaCl, 5.9 mM KCl, 11.6 mM HEPES, 1.5 mM CaCl2, 11.5 mM glucose, 1.2 mM MgCl2, pH 7.3. After permeabilization of the plasma membrane with saponin (10 μg·mL−1, 4 min, 37°C) in Ca2+-free cytosol-like medium (CLM), permeabilized cells were washed (650× g, 2 min) and resuspended (∼107 mL−1) in Mg2+-free CLM containing carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 10 μM) to inhibit mitochondria, and supplemented with CaCl2 to give a final free [Ca2+] of 220 nM after addition of 1.5 mM MgATP. Ca2+-free CLM had the following composition: 2 mM NaCl, 140 mM KCl, 1 mM EGTA, 20 mM PIPES, 2 mM MgCl2, pH 7.0. Permeabilized cells were then distributed into 96-well plates (50 μL, 5 × 105 cells per well), centrifuged (300× g, 2 min) and used for experiments at 20°C. Addition of MgATP (1.5 mM) allowed Ca2+ uptake by the ER, which was monitored at intervals of ∼1 s using a FlexStation-3 plate reader (MDS Analytical Devices, Berkshire, UK; Tovey et al., 2006). After 2 min, when the ER had loaded to steady-state with Ca2+, IP3 was added with CPA (10 μM) to inhibit further Ca2+ uptake. IP3-evoked Ca2+ release was expressed as a fraction of that released by ionomycin (1 μM; Tovey et al., 2006). Similar methods were used to measure IP3-evoked Ca2+ release from intact or permeabilized HEK cells (Tovey et al., 2008). The timings of antagonist additions are described in the figure legends. The affinity of each competitive antagonist (pKD) was determined from the intercept on the abscissa of the Schild plot.

    Concentration–effect relationships were fitted to Hill equations using Prism (version 5.0, GraphPad, San Diego, CA, USA), from which Hill coefficients (h), the fraction of the intracellular Ca2+ stores released by a maximally effective concentrations of IP3, and pEC50 values were calculated.

    Expression of N-terminal fragments of IP3 receptors

    The plasmids used for bacterial expression of GST-tagged N-terminal fragments (NT, residues 1–604) of rat IP3R1, mouse IP3R2 and rat IP3R3 have been described, and their coding sequences have been confirmed (Khan et al., 2013). Plasmids were transformed into BL21-CodonPlus (DE3)-RILP competent cells (Rossi and Taylor, 2011), and grown for 12 h at 37°C in 20 mL of Luria-Bertani (LB) medium containing carbecillin (50 μg·mL−1). The volume of medium was then increased to 1 L, and the incubation was continued at 37°C for 3–4 h until the OD600 reached 1–1.5. Protein expression was induced by addition of IPTG (0.5 mM) for 20 h at 15°C. Bacteria were harvested (6000× g, 5 min), washed twice with cold PBS, and the pellet was suspended (∼109 cells·mL−1) in 50 mL of Tris-EDTA medium (TEM: 50 mM Tris, 1 mM EDTA, pH 8.3) supplemented with 10% PopCulture, 1 mM 2-mercaptoethanol and protease inhibitor cocktail (Roche, Burgess Hill, West Sussex, UK; 1 tablet per 50 mL). After lysis by incubation with lysozyme (100 μg·mL−1) and RNAse (10 μg·mL−1) for 30 min on ice and then sonication (Transsonic T420 water bath sonicator, Camlab, Cambridge, UK; sonicator, 50 Hz, 30 s), the supernatant was recovered (30,000× g, 60 min, 4°C). The supernatant was mixed with glutathione Sepharose 4B beads (50:1, v/v, lysate : beads) and incubated with gentle end-over-end rotation (6 rpm) for 45 min at 4°C. The beads were then loaded onto a PD-10 column and washed twice with PBS and twice with PreScission cleavage buffer (GE Healthcare) supplemented with 1 mM DTT. The column was then incubated with 0.5 mL of PreScission cleavage buffer containing 1 mM DTT and 80 units of GST-tagged PreScission protease for 12 h at 4°C using gentle end-over-end rotation. The PreScission protease cuts an engineered cleavage site to release the NT free of its GST tag. The eluted NT (∼15 mg protein mL−1) was rapidly frozen and stored at −80°C.

    3H-IP3 binding

    Equilibrium competition binding assays were performed at 4°C in 500 μL of CLM (final free [Ca2+] = 220 nM) containing purified NT (30 μg) or cerebellar membranes (5 mg protein), 3H-IP3 (1.5 nM) and appropriate concentrations of competing ligand. Reactions were terminated after 5 min by centrifugation (20,000× g, 5 min) for membranes, or by centrifugation after addition of poly(ethylene glycol)-8000 [30% (w/v), 500 μL] and γ-globulin (30 μL, 25 mg·mL−1) for NT. The pellet was washed (500 μL of 15% PEG or CLM) and solubilized in 200 μL of CLM containing 1% (v/v) Triton-X-100 before liquid scintillation counting. Non-specific binding, whether determined by addition of 10 μM IP3 or by extrapolation of competition curves to infinite IP3 concentration, was <10% of total binding. Results were fitted to Hill equations using Prism, from which IC50 values were calculated. KD (equilibrium dissociation constant) and pKD (–logKD) values were calculated from IC50 values using the Cheng and Prusoff equation (Cheng and Prusoff, 1973).

    Data analysis

    Statistical comparisons used pEC50 (or pKD) values. For paired comparison of the effect of an antagonist, ∆pEC50 values were calculated, where ∆pEC50 = urn:x-wiley:00071188:media:bph12685:bph12685-math-5001. Results are expressed as means ± SEM from n independent experiments. Statistical comparisons used paired Student's t-test or anova followed by Bonferroni's test, with P < 0.05 considered significant.

    Materials

    Sources of many reagents were specified in earlier publications (Rossi et al., 2010a,b; Saleem et al., 2012). IP3 was from Enzo Life Sciences (Exeter, UK). 3H-IP3 (19.3 Ci mmol−1) was from PerkinElmer (Buckinghamshire, UK). Heparin (from porcine mucosa, Mr 5000) and cyclopiazonic acid (CPA) were from Fisher Scientific (Loughborough, UK). Caffeine, 2-APB, lysozyme, RNAse, γ-globulin and poly(ethylene glycol)-8000 were from Sigma-Aldrich (Dorset, UK). Xestopongins C and D were from Calbiochem (Gibbstown, NJ, USA) or isolated and characterized as previously described (Gafni et al., 1997). PopCulture was from Novagen (Darmstadt, Germany). Simply Blue stain was from Invitrogen (Renfrewshire, Scotland). Dioxin-free isopropyl-β-D-thiogalactoside (IPTG), and Luria–Bertani agar and broth were from Formedium (Norfolk, UK). Glutathione Sepharose 4B beads and GST-tagged PreScission protease were from GE Healthcare (Buckinghamshire, UK). Carbecillin was from Melford Laboratories (Suffolk, UK). BL21-CodonPlus (DE3)-RILP competent bacteria were from Agilent Technology (Berkshire, UK).

    Results

    Heparin is a competitive antagonist with different affinities for IP3 receptor subtypes

    Heparin is a competitive antagonist of IP3-evoked Ca2+ release (Ghosh et al., 1988), but it is membrane-impermeable and it has many additional effects. These include uncoupling of receptors from G-proteins (Willuweit and Aktories, 1988; Dasso and Taylor, 1991), stimulation of ryanodine receptors (Ehrlich et al., 1994) and inhibition of IP3 3-kinase (Guillemette et al., 1989). To assess the effects of heparin on each IP3R subtype, permeabilized DT40 cells expressing each of the three IP3R subtypes were incubated with heparin for 35 s. The effect of IP3 on Ca2+ release from the intracellular stores was then assessed (Figure 1A). In permeabilized DT40-IP3R1 cells, heparin caused parallel rightward shifts of the concentration–response relationship for IP3-evoked Ca2+ release (Figure 1B). Schild plots, which had slopes of 0.95 ± 0.02 (mean ± SEM, n = 3), established that the equilibrium dissociation constant (KD) for heparin was 4.1 μg·mL−1 (pKD = 5.39 ± 0.00) (Figure 1C). Similar results were obtained when adenophostin A (AdA), a high-affinity agonist of IP3Rs (Rossi et al., 2010b; Saleem et al., 2013), was used to stimulate Ca2+ release. The Schild plots had slopes of 0.94 ± 0.03 (n = 3) and the KD for heparin was 6.9 μg·mL−1 (pKD = 5.16 ± 0.05) (Figure 1D and E; Table 1).

    figure

    Heparin competitively inhibits IP3-evoked Ca2+ release via type 1 IP3 receptors. (A) Typical traces from a population of permeabilized DT40-IP3R1 cells showing the fluorescence (RFU, relative fluorescence units) recorded from a luminal Ca2+ indicator after addition of MgATP (1.5 mM), heparin (400 μg·mL−1, red lines; or CLM alone, black lines) and then IP3 (1 or 100 μM). The traces show average responses from two wells in a single plate. (B) Experiments similar to those in A show concentration-dependent effects of IP3 on Ca2+ release in the presence of the indicated concentrations of heparin. (C) Schild analysis of the results shown in B. (D, E) Similar analyses of the effects of heparin on AdA-evoked Ca2+ release via IP3R1. Results (B–E) are means ± SEM from three experiments.

    Table 1. Effects of heparin on IP3-evoked Ca2+ release and IP3 binding
    Functional analysis aBinding pEC50(IP3)-pKD(heparin)
    IP3 or AdA heparin heparin
    pEC50 pKD pKD
    IP3R1 IP3 7.47 ± 0.02 5.39 ± 0.00 4.66 2.08 ± 0.02
    IP3R1 AdA 8.35 ± 0.03 5.16 ± 0.05
    IP3R2 IP3 6.82 ± 0.04 4.66 ± 0.07 4.62 2.16 ± 0.09*
    IP3R3 IP3 6.66 ± 0.07 5.55 ± 0.09 5.34 1.11 ± 0.08*
    IP3R3 AdA 7.71 ± 0.01 5.68 ± 0.04
    • From experiments similar to those shown in Figures 1 and 2, AdA or IP3-evoked Ca2+ release and their sensitivity to heparin were used to determine pEC50 (as M) and pKD (as g mL−1) for DT40 cells expressing IP3R1, IP3R2 or IP3R3. Results are means ± SEM from three independent experiments (six for IP3R3).
    • a The affinities for heparin determined from equilibrium-competition binding with 3H-IP3 to Sf9 membranes expressing IP3R1-3 are reproduced from (Nerou et al., 2001). The batch of heparin used for those binding studies was different from that used for the work reported here. The final column (derived from the results shown in Figures 1B,C and 2A–D) shows paired comparisons of pEC50(IP3) – pKD(heparin) as a means of reporting the relative effectiveness with which heparin might be expected to block IP3-evoked Ca2+ release via different IP3R subtypes. The results suggest that IP3R3 is likely to be substantially more susceptible to inhibition than IP3R1 or IP3R2.
    • *Denotes a value significantly different from IP3R1 in the final column (P < 0.05).
    figure

    Heparin is a competitive antagonist with different affinities for types 2 and 3 IP3 receptors. (A) Concentration-dependent release of Ca2+ by IP3 from the intracellular stores of DT40-IP3R2 cells in the presence of the indicated concentrations of heparin added 35 s before IP3. (B) Schild plot of the results. (C–F) Similar analyses of DT40-IP3R3 cells stimulated with IP3 (C, D) or AdA (E, F). For D, where maximal attainable concentrations of IP3 were insufficient to evoke maximal responses in the presence of the highest concentrations of heparin, the Schild plot shows dose ratios calculated from IP3 concentrations that evoked 40% Ca2+ release. Results (A–F) are mean ± SEM from three experiments.

    A similar analysis of the effects of heparin on IP3-evoked Ca2+ release from permeabilized DT40-IP3R2 cells was also consistent with competitive antagonism. The slope of the Schild plots was 0.97 ± 0.06 (n = 3) and the KD for heparin was 22 μg·mL−1 (pKD = 4.66 ± 0.07) ( Figure 2A and B). IP3R3 are less sensitive to IP3 than the other subtypes (Iwai et al., 2007; Saleem et al., 2013) (Table 1). This made it difficult to add IP3 at concentrations sufficient to achieve maximal Ca2+ release in the presence of heparin concentrations greater than 5 μg·mL–1 (Figure 2C). Assuming the maximal response to IP3 was unaffected by heparin, we used the concentrations of IP3 that evoked release of 40% of the intracellular stores to construct Schild plots for IP3R3. The results were consistent with competitive antagonism. The slope of the Schild plots was 1.14 ± 0.41 (n = 3) and the KD for heparin was 2.8 μg·mL−1 (pKD = 5.55 ± 0.09) (Figure 2D and Table 1). AdA has ∼10-fold higher affinity than IP3 for all three IP3R subtypes (Table 1) (Rossi et al., 2010a; Saleem et al., 2013), and we have shown that the affinity of heparin for IP3R1 is similar whether IP3 or AdA is used to evoke Ca2+ release (Figure 1B–E). To obtain an independent measure of the affinity of IP3R3 for heparin, free of the problems associated with using IP3, we therefore repeated the Schild analysis using AdA to stimulate Ca2+ release. These conditions provided complete concentration–effect relationships for AdA at a wider range of heparin concentrations (Figure 2E). The Schild plots had a slope of 0.98 ± 0.04 (n = 6) and the KD for heparin was 2.1 μg·mL−1 (pKD = 5.68 ± 0.04) (Figure 2F and Table 1). The affinity of heparin for IP3R3 was therefore similar whether measured using IP3 or AdA to evoke Ca2+ release.

    These functional analyses establish that heparin is a competitive antagonist of IP3 at all three IP3R subtypes, but with different affinities for each (IP3R3 > IP3R1 ≥ IP3R2) (Table 1). The results are consistent with an analysis of IP3 binding to mammalian IP3R expressed in Sf9 cells (Nerou et al., 2001), where the pKD values and rank order of heparin affinity (IP3R3 > IP3R1 ∼ IP3R2) were similar to those from the present functional analyses (Table 1).

    Heparin binding is not solely determined by its interactions with the IP3-binding site

    Activation of IP3Rs is initiated by binding of IP3 to the IP3-binding core (IBC, residues 224-604 of IP3R1) within the N-terminal region of each IP3R subunit (see Introduction) (Seo et al., 2012). The only contacts between IP3 and the IP3R are mediated by residues within the IBC (Bosanac et al., 2002), but interaction of the N-terminal suppressor domain (residues 1-223) with the IBC reduces its affinity for IP3. Hence, the IBCs from different IP3R subtypes bind IP3 with similar affinity, whereas the larger N-terminal regions (NT, residues 1-604) have lower affinities that differ between subtypes. The NTs bind IP3 with two- to threefold greater affinities than those of full-length IP3Rs, but the NTs and full-length IP3Rs have the same rank order of affinities for IP3 (NT2 > NT1 > NT3) (Iwai et al., 2007; Rossi et al., 2009). The results shown in Figure 3A and B, which show IP3 binding to bacterially expressed NTs from each of the three IP3R subtypes (NT1-3), confirm previous results. Surprisingly, however, equilibrium-competition binding of heparin to NTs in medium that matches that used to measure IP3-evoked Ca2+ release was not consistent with the results obtained from functional analyses (Figure 3C). The affinity of the NT for heparin was up to 2000-fold greater than that measured in functional analyses, and the rank order of affinity for heparin was different for NTs (NT2 > NT1 > > NT3) and full-length IP3Rs (IP3R3 > IP3R1 ≥ IP3R2) (Nerou et al., 2001; Tables 1 and 2).

    figure

    Heparin binding is not solely determined by its interactions with the IP3-binding core. (A) Immunoblots of purified NT1-3 (∼15 μL protein per lane) using an antiserum that recognizes a conserved sequence within all three IP3R subtypes (residues 62–75 in rat IP3R1). The positions of Mr markers (kDa) are shown alongside each blot. (B, C) Equilibrium-competition binding of IP3 (B) and heparin (C) to purified NT1-3 in CLM. (D, E) Similar analyses of binding to cerebellar membranes (IP3R1). Results (B–E) are means ± SEM from three to six experiments.

    Table 2. Heparin and IP3 binding to N-terminal fragments of IP3 receptor subtypes
    NT1 NT2 NT3 IP3R1
    IP3 7.76 ± 0.07 8.67 ± 0.15 7.39 ± 0.08 7.13 ± 0.08
    Heparin 7.42 ± 0.09 7.95 ± 0.32 6.59 ± 0.09* 5.61 ± 0.13
    • Equilibrium-competition binding with 3H-IP3 was used to measure pKD values for IP3 (as M) and heparin (as g mL−1) binding to purified NT1-3 and cerebellar membranes (IP3R1). Results are means ± SEM from three to six experiments.
    • *Denotes a significant difference from NT1 (P < 0.05) for urn:x-wiley:00071188:media:bph12685:bph12685-math-5002.

    IP3R1 is the major (>99%) subtype in cerebellar membranes (Wojcikiewicz, 1995). Equilibrium-competition binding of heparin to cerebellar membranes in CLM established that the affinity of IP3R1 for heparin (pKD = 5.61 ± 0.13, n = 3) was similar to that derived from Schild analysis of DT40-IP3R1 cells (pKD = 5.39 ± 0.00, n = 3) and similar to that reported for heparin binding to IP3R1 heterologously expressed in Sf9 cells (Nerou et al., 2001), but very different to the heparin affinity of NT1 (pKD = 7.42 ± 0.09, n = 3) (Tables 1 and 2). These results demonstrate that the IBC is not the only determinant of competitive heparin binding to IP3Rs and suggest either that access of heparin to the IBC is influenced by additional interactions or that heparin binding to an additional site affects IP3R gating.

    2-APB selectively inhibits Ca2+ release via type 1 IP3 receptors without affecting IP3 binding

    2-APB is membrane-permeant and is often used to inhibit IP3-evoked Ca2+ release (Maruyama et al., 1997; Missiaen et al., 2001; Bilmen et al., 2002), but it has many additional effects. These include modulation of store-operated Ca2+ entry (Goto et al., 2010) and inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) that mediates Ca2+ sequestration by the ER (Missiaen et al., 2001; Bilmen et al., 2002; Bultynck et al., 2003). In permeabilized DT40-IP3R1 cells, 50 μM 2-APB had no effect on Ca2+ uptake by the ER, although higher concentrations reduced the steady-state Ca2+ content (Figure 4A and B). This is consistent with high concentrations of 2-APB causing inhibition of SERCA.

    figure

    2-APB selectively inhibits Ca2+ release via type 1 IP3 receptors. (A) Ca2+ uptake into the intracellular stores of permeabilized DT40-IP3R1 cells is shown after addition of ATP in the presence of the indicated concentrations of 2-APB. Each trace is the average from two wells in a single plate. (B) Summary results show effects of 2-APB on Ca2+ contents measured 180 s after addition of ATP. (C–E) Concentration-dependent effects of IP3 on Ca2+ release from permeabilized DT40-IP3R1-3 cells alone or with the indicated concentrations of 2-APB added 35 s before IP3. (F) Binding of 3H-IP3 (1.5 nM) to cerebellar membranes (IP3R1), with 3 μM IP3 (non-specific) or with 2-APB. Results (B–F) are means ± SEM from three to nine experiments. *P < 0.05, significantly different from control.

    In permeabilized DT40-IP3R1 cells, 2-APB caused a concentration-dependent inhibition of IP3-evoked Ca2+ release (Figure 4C). With 50 μM 2-APB, the highest concentration that avoids inhibition of Ca2+ uptake, there was an approximately sevenfold decrease in IP3 sensitivity (ΔpEC50 = 0.84 ± 0.12) with no effect on the maximal response to IP3 (Figure 4C). The same concentration of 2-APB (50 μM) had no significant effect on IP3-evoked Ca2+ release from permeabilized DT40-IP3R2 or DT40-IP3R3 cells (Figure 4D and E). When the 2-APB concentration was increased to 100 μM, which caused some inhibition of Ca2+ uptake (Figure 4A and B), there was some inhibition of IP3R3, but no effect on IP3-evoked Ca2+ release via IP3R2 (Figure 4D and E; Table 3).

    Table 3. Selective inhibition of IP3 receptor subtypes by common antagonists
    IP3R1 IP3R2 IP3R3
    ΔpEC50 (M) ΔMax (%) ΔpEC50 (M) ΔMax (%) ΔpEC50 (M) ΔMax (%)
    Heparin, 400 μg·mL−1 1.88 ± 0.05* −7 ± 2 ND 2.34 ± 0.07* −3 ± 3
    Heparin, 800 μg·mL−1 ND 1.49 ± 0.09* −4 ± 2 ND
    Caffeine, 70 mM 0.61 ± 0.07* 12 ± 4 −0.2 ± 0.07 −1 ± 0 −0.07 ± 0.08 0 ± 5
    2-APB, 50 μM 0.84 ± 0.12* 0 ± 4 −0.05 ± 0.10 0 ± 4 0.02 ± 0.09 8 ± 4
    Xestospongin C, 20 μM 0.21 ± 0.10* 6 ± 2* −0.06 ± 0.04 1 ± 1 0.12 ± 0.03* 1 ± 2
    Xestospongin D, 20 μM 0.26 ± 0.09* 18 ± 2* −0.15 ± 0.05 8 ± 3* 0.21 ± 0.10* 2 ± 2
    • Summary of the functional analyses of antagonists on IP3-evoked Ca2+ release from permeabilized DT40-IP3R1-3 cells. The pEC50 values for IP3 and the maximal Ca2+ release are each expressed relative to the response evoked in paired controls without antagonist (Δ = control – response with antagonist). A positive Δ value demonstrates an inhibition of IP3-evoked Ca2+ release by the antagonist. The results with Xestospongins C and D are pooled from experiments that included pre-incubation periods of 7 and 12 min (see Supporting Information Table S1). Results are means ± SEM from three to nine experiments.
    • *Denotes a value significantly greater than 0 (P < 0.025, one-tailed test).
    • ND, not determined.

    Binding of 3H-IP3 to IP3R1 of cerebellar membranes in CLM was unaffected by 2-APB (Figure 4F) consistent with published results (Maruyama et al., 1997; Bilmen et al., 2002). This demonstrates that inhibition of IP3R1 by 2-APB is neither due to competition with IP3 nor to allosteric inhibition of IP3 binding.

    Caffeine is a low-affinity antagonist of type 1 IP3 receptors

    Caffeine is another membrane-permeant antagonist of IP3-evoked Ca2+ release (Parker and Ivorra, 1991; Brown et al., 1992; Bultynck et al., 2003; Laude et al., 2005), but it is effective only at high (mM) concentrations and it has many additional effects (Michelangeli et al., 1995; Taylor and Tovey, 2010). These include stimulation of ryanodine receptors, inhibition of cyclic nucleotide phosphodiesterases, competitive antagonism of adenosine receptors, and effects on the fluorescence of some Ca2+ indicators (Brown et al., 1992; Ehrlich et al., 1994; Michelangeli et al., 1995; McKemy et al., 2000; Taylor and Tovey, 2010). High concentrations of caffeine (10–70 mM) inhibited Ca2+ release via IP3R1 (Figure 5A) without affecting 3H-IP3 binding to cerebellar membranes (Figure 5D). The latter is consistent with published work (Brown et al., 1992). The maximal attainable concentration of caffeine (70 mM) caused an approximately fourfold decrease in IP3 sensitivity (ΔpEC50 = 0.61 ± 0.07) (Figure 5A). Caffeine had no significant effect on IP3-evoked Ca2+ release via IP3R2 or IP3R3 (Figure 5B and C; Table 3). At the highest concentration used (70 mM), caffeine significantly reduced the Ca2+ content of the intracellular stores, but this inhibition was similar for DT40 cells expressing each of the IP3R subtypes (Figure 5E). Inhibition of Ca2+ sequestration by the ER is unlikely, therefore, to account for the selective inhibition of IP3-evoked Ca2+ release via IP3R1 (Table 3). These results demonstrate that a high concentration of caffeine modestly, but selectively, inhibits IP3-evoked Ca2+ release via IP3R1 without affecting IP3 binding.

    figure

    Caffeine is a low-affinity antagonist of type 1 IP3R receptors. (A–C) Concentration-dependent effects of IP3 on Ca2+ release from permeabilized DT40-IP3R1-3 cells in the presence of the indicated concentrations of caffeine added 4 min before IP3. (D) Binding of 3H-IP3 (1.5 nM) to cerebellar membranes alone (total), with 3 μM IP3 (non-specific) or caffeine. (E) Effect of caffeine added 2 min before ATP on the steady-state Ca2+ content of the intracellular stores (percentage of matched control cells) measured 90 s after addition of ATP to DT40-IP3R1-3 cells. Results (A–E) are means ± SEM from three experiments. *P < 0.05 significantly different from control.

    Xestospongins do not effectively inhibit IP3-evoked Ca2+ release

    Xestospongin C is membrane-permeant and was reported to inhibit IP3-evoked Ca2+ release from cerebellar microsomes (IC50 = 358 nM) without affecting IP3 binding (Gafni et al., 1997). Xestospongin D is less potent. Higher concentrations of Xestospongin C (10–20 μM) were required to inhibit IP3-evoked Ca2+ release in intact cells. We assessed the effects of Xestospongins C and D from different suppliers (see Materials) on Ca2+ release mediated by each of the three IP3R subtypes.

    Pre-incubation of permeabilized DT40 cells with Xestospongin C (5–20 μM from either source) for 5–12 min before addition of IP3 had no significant effect on IP3-evoked Ca2+ release mediated by any of the three IP3R subtypes (Supporting Information Table S1). Figure 6A–C show IP3-evoked Ca2+ release after a 5 min pre-incubation with 5 μM purified Xestospongin C (Gafni et al., 1997). It had no significant effect on either the response to IP3 (Figure 6A–C) or the Ca2+ content of the stores (Figure 6D). Pooling all experiments with the highest concentration of Xestospongin C (20 μM, n = 6) revealed a statistically significant (P < 0.025, one-tailed test), but very small, inhibition of the maximal response from IP3R1, and an even smaller increase in pEC50 for IP3R1 and IP3R3 (Table 3 and Supporting Information Table S1).

    figure

    Xestospongins do not effectively inhibit IP3 receptors. (A–C) IP3-evoked Ca2+ release from permeabilized DT40-IP3R1-3 cells is shown with or without 5 μM Xestospongin C (from Gafni et al., 1997) added 5 min before IP3. (D) Effects of Xestospongin C (5–20 μM) added 5–12 min before ATP on the Ca2+ content of the intracellular stores (percentages of matched controls without Xestospongin). (E–H) Similar analyses using Xestospongin D (10 μM added 5 min before IP3). Results (A–H) are means ± SEM from three experiments.

    Similar treatments with Xestospongin D (10–20 μM from either source) for 5–12 min caused a modest, but statistically significant (P < 0.025, one-tailed test), inhibition of IP3-evoked Ca2+ release via IP3R1 (Supporting Information Table S1). Figure 6E–H show that a 5 min pre-incubation with 10 μM purified Xestospongin D (Gafni et al., 1997) had no effect on the Ca2+ content of the intracellular stores, but modestly inhibited IP3-evoked Ca2+ release via IP3R1 (P < 0.025, one-tailed test, Figure 6E). Pooling results with the highest concentration of Xestospongin D (20 μM, n = 6) revealed a statistically significant (P < 0.025, one-tailed test), but very small, inhibition of the maximal response from IP3R1 and IP3R2, and a tiny increase in the pEC50 for IP3R1 and IP3R3 (Table 3 and Supporting Information Table S1). These small inhibitory effects of Xestospongins C and D are not sufficient to be useful, and nor are they sufficient to reliably assess whether there is any subtype-selective interaction of Xestospongins with IP3Rs.

    We also assessed the effects of Xestospongins on IP3-evoked Ca2+ release from intact and permeabilized HEK cells. IP3 caused a concentration-dependent release of Ca2+ from the intracellular stores of permeabilized HEK cells (Figure 7A and B). Pre-incubation of the permeabilized cells for 5 min with Xestospongin C (5 μM) or Xestospongin D (10 μM) had no effect on the Ca2+ content of the intracellular stores (Figure 7C) or the Ca2+ release evoked by IP3 (Figure 7A and B). Carbachol, via endogenous M3 muscarinic receptors of HEK cells, stimulates PLC and thereby IP3-evoked Ca2+ release. Preincubation of HEK cells with Xestospongin C or D (10 μM) for 30 min had no significant effect on the Ca2+ signals evoked by any concentration of carbachol (Figure 7D). This conflicts with published results from similar experiments, where Xestospongin C (10 μM for 30 min) caused substantial, though incomplete, inhibition of carbachol-evoked Ca2+ signals (Kurian et al., 2009). It is, however noteworthy, in light of evidence that Xestospongins have been reported to inhibit Ca2+ uptake into the ER (Castonguay and Robitaille, 2002; Solovyova et al., 2002), that in the experiments from Kurian et al. HEK cells were incubated with Xestospongin for 30 min in Ca2+-free medium, while in our experiments extracellular free Ca2+ was removed immediately before stimulation with carbachol. The discrepant results may, therefore, reflect an increased loss of Ca2+ from intracellular stores during prolonged exposure to Xestospongin in Ca2+-free medium.

    figure

    Xestospongins do not inhibit IP3-evoked Ca2+ signals in HEK cells. (A–C) Permeabilized HEK cells were incubated with Xestospongin C (5 μM) or Xestospongin D (10 μM) for 5 min before addition of IP3. Both Xestospongins were prepared as described (Gafni et al., 1997). Results show IP3-evoked Ca2+ release (A, B) or the steady-state Ca2+ content of the intracellular stores (C, as a percentage of matched controls without Xestospongin). (D) Concentration-dependent effects of carbachol on the increase in intracellular free Ca2+ concentration [Ca2+]i of intact fluo-4-loaded HEK cells after treatment with Xestospongins C or D (10 μM for 30 min). pEC50 (M) values for the carbachol-evoked Ca2+ signals were 4.99 ± 0.13, 4.92 ± 0.23 and 4.70 ± 0.11 for control cells and cells treated with Xestospongins C and D respectively. Results (A–D) are means ± SEM. from three experiments.

    Discussion

    Acute analyses of IP3-evoked Ca2+ signalling are handicapped by lack of effective and selective antagonists (Michelangeli et al., 1995; Bultynck et al., 2003). Furthermore, the subtype-selectivity and in many cases the mechanism of action of the antagonists that are routinely used are not known. We have addressed these issues by examining the functional effects of the most widely used antagonists of IP3R in cells expressing only a single IP3R subtype.

    Heparin is a competitive antagonist of IP3 at cerebellar IP3Rs (Ghosh et al., 1988), most likely because as a polyanion it may partially mimic the phosphate groups of IP3. That is consistent with evidence that other polyanions, like decavanadate, ATP and dextran sulphate, can also competitively inhibit IP3Rs (Bultynck et al., 2003). Our functional analyses establish that heparin is a competitive antagonist of all three IP3R subtypes, but with modestly different affinities for each (IP3R3 > IP3R1 ≥ IP3R2) (Figures 1 and 2; Table 1). The affinities of IP3R subtypes for heparin derived from functional analyses were similar to those determined from equilibrium-competition binding to native IP3R1 (Figure 3E) or to heterologously expressed IP3R subtypes (Table 1). However, heparin bound to N-terminal fragments (NT) of IP3Rs that include the IBC with an affinity that was up to 2000-fold greater than its affinity for the corresponding full-length IP3R (Tables 1 and 2). Furthermore, the rank order of heparin affinity for IP3R1-3 and NT1-3 was different. We conclude that heparin inhibits IP3-evoked Ca2+ release by competing with IP3, but its access to the IBC is substantially impaired in full-length IP3Rs within native membranes. Phospholipids may contribute to the substantially lesser affinity of heparin for IP3R in native membranes by electrostatically repelling the approach of polyanionic heparin to the membrane-bound IBC. In addition, we suggest that charged residues on the IP3R surface may differentially influence heparin access to the IBC of each IP3R subtype and thereby contribute to the modestly different affinities of heparin for IP3R subtypes (Table 1). Our observations have more general significance for analyses of competitive antagonism. We have demonstrated that properties of either the receptor or its environment that are remote from the ligand-binding site may significantly affect the apparent affinity of a receptor for a competitive antagonist.

    Because heparin is a competitive antagonist of IP3 (Figures 1 and 2), its experimental utility will depend on its affinity relative to IP3 for each IP3R subtype. Table 1 addresses this issue by comparing measured affinities for heparin with EC50 values for IP3 as an estimate of the relative affinity of each IP3R subtype for IP3. The analysis indicates that within native cells, responses of IP3R3 to IP3 are likely to be more susceptible to inhibition by heparin than the responses mediated by other IP3R subtypes.

    Both 2-APB and caffeine selectively inhibited IP3-evoked Ca2+ release via IP3R1, without affecting IP3 binding (Figures 4 and 5; Table 3). Higher concentrations of 2-APB caused some inhibition of IP3R3, but this was accompanied by inhibition of ER Ca2+ uptake (Figure 4). The highest concentration of caffeine used (70 mM) also inhibited Ca2+ sequestration by the ER, but without significantly affecting the sensitivity to IP3 of IP3R2 or IP3R3, or the fraction of the remaining Ca2+ stores released via them by a maximally effective concentration of IP3 (Figure 5). Previous analyses of cells expressing different mixtures of native IP3R subtypes have also suggested that IP3R2 may be resistant to inhibition by 2-APB (Gregory et al., 2001; Hauser et al., 2001; Kukkonen et al., 2001; Bootman et al., 2002; Soulsby and Wojcikiewicz, 2002) and caffeine (Kang et al., 2010). The mechanism of action of 2-APB is unresolved, but for IP3R1 caffeine appears to compete with ATP for the site through which ATP potentiates IP3-evoked Ca2+ release (Missiaen et al., 1994; Maes et al., 2001). This mechanism appears not to explain the actions of 2-APB (Missiaen et al., 2001). ATP potentiates IP3-evoked Ca2+ release via all three IP3R subtypes (Smith et al., 1985; Mak et al., 1999; Maes et al., 2001; Tu et al., 2005; Betzenhauser et al., 2008), but the mechanisms and ATP-binding sites differ (Betzenhauser et al., 2008; 2009; Betzenhauser and Yule, 2010). Work from Yule and his colleagues suggests that IP3R2 is most sensitive to ATP and for it, but not other IP3R subtypes, an ATPB site within each IP3R subunit mediates the potentiating effect of ATP (Betzenhauser and Yule, 2010). It is, therefore, tempting to speculate that the different sensitivities of IP3R subtypes to inhibition by caffeine (Figure 5) may be related to their different modes of regulation by ATP.

    Xestospongins were initially shown to inhibit IP3-evoked Ca2+ release selectively (Gafni et al., 1997), and numerous subsequent analyses of their effects on intact cells are consistent with inhibition of IP3Rs (e.g. Bishara et al., 2002; Duncan et al., 2007; Oka et al., 2002; Ozaki et al., 2002; Rosado and Sage, 2000; Schafer et al., 2001; Yuan et al., 2005), but few of these later analyses directly addressed the effects of Xestospongins on IP3Rs (e.g. Oka et al., 2002; Ozaki et al., 2002). The latter is important because Xestospongins have additional effects that include inhibition of SERCA (De Smet et al., 1999; Castonguay and Robitaille, 2002; Solovyova et al., 2002), store-operated Ca2+ entry (Bishara et al., 2002), L-type Ca2+ channels and Ca2+-activated K+ channels (Ozaki et al., 2002), and modulation of ryanodine receptors (Ta et al., 2006). The potencies of Xestospongins also differ between studies and some reports challenge whether they effectively inhibit IP3Rs (Solovyova et al., 2002; Duncan et al., 2007; Govindan and Taylor, 2012). We used two sources of Xestospongins C and D, a range of concentrations and incubation periods, two different cell types (see also Govindan and Taylor, 2012), and both intact and permeabilized cells. Although the Xestospongins caused some inhibition of IP3-evoked Ca2+ release, none of our analyses succeeded in demonstrating that attainable (≤20 μM) concentrations of Xestospongins substantially inhibited any IP3R subtype (Figures 6 and 7; Table 3; Supporting Information Table S1).

    We conclude that none of the commonly used antagonists of IP3Rs is free of pitfalls. Heparin is perhaps the most reliable, it is competitive with IP3, but it is membrane-impermeant, and its binding to the IBC of IP3Rs is influenced by more distant residues that cause it to bind with different affinity to each IP3R subtype (Figures 1-3). Caffeine and 2-APB are membrane-permeant, they do not compete with IP3, but neither achieves effective inhibition of IP3Rs without affecting other Ca2+-regulating proteins, and both show selectivity for IP3R1 (Figures 4 and 5). Xestospongins are membrane-permeant and reported to inhibit IP3-evoked Ca2+ release without affecting IP3 binding (Gafni et al., 1997), but in our hands they do not inhibit any IP3R subtype (Figures 6 and 7).

    Acknowledgements

    Supported by the Wellcome Trust (101844), Biotechnology and Biological Sciences Research Council (BB/H009736) and a studentship from the Jameel Family Trust to Huma Saleem. We thank Dr Ana Rossi for providing plasmids.

      Author contributions

      HS performed and analysed experiments. TFM provided reagents. CWT and SCT supervised the project and contributed to analysis. CWT wrote the paper. All authors reviewed the paper.

      Conflict of interest

      None