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

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.


Introduction
Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca 2+ channels expressed in the membranes of the endoplas-mic 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 Ca 2+ signals triggered by IP3-evoked Ca 2+ 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 Ca 2+ -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 Ca 2+ 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 Ca 2+ (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; 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 Ca 2+ 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 Ca 2+ 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 membranepermeable. 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 Ca 2+ 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 IP 3R 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 Ca 2+ release via each IP3R subtype.
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 Ca 2+ stores released by a maximally effective concentrations of IP3, and pEC50 values were calculated.

Expression of N-terminal fragments of IP 3 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 (∼10 9 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-overend 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.

H-IP binding
Equilibrium competition binding assays were performed at 4°C in 500 μL of CLM (final free [Ca 2+ ] = 220 nM) containing purified NT (30 μg) or cerebellar membranes (5 mg protein), 3 H-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). 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.

Heparin is a competitive antagonist with different affinities for IP 3 receptor subtypes
Heparin is a competitive antagonist of IP3-evoked Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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). A similar analysis of the effects of heparin on IP3-evoked Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ release. These conditions provided complete concentrationeffect 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 Ca 2+ 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).

Figure 1
Heparin competitively inhibits IP3-evoked Ca 2+ release via type 1 IP3 receptors. 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 IP 3 -binding site
Activation of IP3Rs is initiated by binding of IP3 to the IP3binding 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 Ca 2+ 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).
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 The affinities for heparin determined from equilibrium-competition binding with 3 H-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 Ca 2+ 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). Equilibrium-competition binding with 3 H-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 pK D heparin .

Figure 2
Heparin is a competitive antagonist with different affinities for types 2 and 3 IP3 receptors. 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.
Binding of 3 H-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 IP 3 receptors
Caffeine is another membrane-permeant antagonist of IP3evoked Ca 2+ 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 Ca 2+ 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 Ca 2+ release via IP3R1 ( Figure 5A  ( 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 Ca 2+ release via IP3R2 or IP3R3 (Figure 5B and C; Table 3). At the highest concentration used (70 mM), caffeine significantly reduced the Ca 2+ content of the intracellular stores, but this inhibition was similar for DT40 cells expressing each of the IP3R subtypes ( Figure 5E). Inhibition of Ca 2+ sequestration by the ER is unlikely, therefore, to account for the selective inhibition of IP3-evoked Ca 2+ release via IP3R1 (Table 3). These results demonstrate that a high concentration of caffeine modestly, but selectively, inhibits IP3-evoked Ca 2+ release via IP3R1 without affecting IP3 binding.

Xestospongins do not effectively inhibit IP 3 -evoked Ca 2+ release
Xestospongin C is membrane-permeant and was reported to inhibit IP3-evoked Ca 2+ 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 IP3evoked Ca 2+ release in intact cells. We assessed the effects of Xestospongins C and D from different suppliers (see Materials) on Ca 2+ 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 Ca 2+ release mediated by any of the three IP3R subtypes (Supporting Information Table S1). Figure 6A-C show IP3-evoked Ca 2+ 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 Ca 2+ 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 IP 3R1, and an even smaller increase in pEC50 for IP3R1 and IP3R3 (Table 3 and Supporting Information Table S1).
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 IP3evoked Ca 2+ 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 Ca 2+ content of the intracellular stores, but modestly inhibited IP3-evoked Ca 2+ 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.
light of evidence that Xestospongins have been reported to inhibit Ca 2+ 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 Ca 2+ -free medium, while in our experiments extracellular free Ca 2+ was removed immediately before stimulation with carbachol. The discrepant results may, therefore, reflect an increased loss of Ca 2+ from intracellular stores during prolonged exposure to Xestospongin in Ca 2+ -free medium.

Discussion
Acute analyses of IP3-evoked Ca 2+ 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 equilibriumcompetition binding to native IP3R1 ( Figure 3E) or to heter-

Figure 5
Caffeine is a low-affinity antagonist of type 1 IP3R receptors. (A-C) Concentration-dependent effects of IP3 on Ca 2+ release from permeabilized DT40-IP3R1-3 cells in the presence of the indicated concentrations of caffeine added 4 min before IP3. (D) Binding of 3 H-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 Ca 2+ 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. ologously 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 IP3evoked Ca 2+ 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 substan-tially 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 ligandbinding 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 Ca 2+ 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 Ca 2+ uptake (Figure 4). The highest concentration of caffeine used (70 mM) also inhibited Ca 2+ sequestration by the ER, but without significantly affecting the sensitivity to IP3 of IP3R2 or IP3R3, or the fraction of the remaining Ca 2+ 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 IP3evoked Ca 2+ release (Missiaen et al., 1994;Maes et al., 2001). This mechanism appears not to explain the actions of 2-APB . ATP potentiates IP3-evoked Ca 2+ 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;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.

Figure 7
Xestospongins do not inhibit IP3-evoked Ca 2+ 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 Ca 2+ release (A, B) or the steady-state Ca 2+ 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 Ca 2+ concentration [Ca 2+ ]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 Ca 2+ 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. additional effects that include inhibition of SERCA (De Smet et al., 1999;Castonguay and Robitaille, 2002;Solovyova et al., 2002), store-operated Ca 2+ entry (Bishara et al., 2002), L-type Ca 2+ channels and Ca 2+ -activated K + channels , 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 Ca 2+ 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 membraneimpermeant, 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 Ca 2+ -regulating proteins, and both show selectivity for IP3R1 (Figures 4 and 5). Xestospongins are membrane-permeant and reported to inhibit IP3-evoked Ca 2+ release without affecting IP3 binding (Gafni et al., 1997), but in our hands they do not inhibit any IP3R subtype (Figures 6  and 7).