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The N-terminal Ca²⁺-Independent Calmodulin-Binding Site on the Inositol 1,4,5-trisphosphate Receptor Is Responsible for Calmodulin Inhibition, Even Though This Inhibition Requires Ca²⁺

Laboratorium voor Fysiologie, Katholieke Universiteit Leuven Campus Gasthuisberg O/N, Leuven, Belgium (N.N.K., G.B., K.S., J.B.P., G.C., L.M., H.d.S.); Molecular and Cellular Biology Program and Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts (J.S., R.A.F.); and Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, Tokyo, Japan (K.M.)

Received February 25, 2004; accepted April 18, 2004

	Abstract

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Calmodulin (CaM) is a ubiquitous Ca²⁺-sensor protein that plays an important role in regulating a large number of Ca²⁺ channels, including the inositol 1,4,5-trisphosphate receptor (IP₃R). CaM binds to the IP₃R at Ca²⁺-dependent as well as at Ca²⁺-independent interaction sites. In this study, we have investigated the Ca²⁺-independent CaM-binding site for its role in the regulation of the Ca²⁺-dependent bell-shaped activation curve of the IP₃R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca²⁺. Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on IP₃R1. By interacting with the N-terminal Ca²⁺-independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition of IP₃ binding. Therefore, suramin also potently inhibited IP₃-induced Ca²⁺ release (IICR) from permeabilized cells predominantly expressing IP₃R1 (L15 fibroblasts) or IP₃R3 (Lvec fibroblasts), even though the IP₃R3 does not contain Ca²⁺-dependent CaM-binding sites. Furthermore, we have found that CaM₁₂₃₄, a CaM mutated in its four EF hands, inhibited IICR in a Ca²⁺-dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition requires Ca²⁺ but CaM itself is not the Ca²⁺ sensor for the inhibition of the IP₃R.

In this study, we have investigated the functional effects of CaM on the IP₃R, particularly the role of its N-terminal CaM-binding site. We have used suramin, a polysulfonated napthylurea, to investigate the molecular mechanism of the inhibition of the IP₃R by CaM. Suramin has been shown to interact in a direct way with different types of CaM-binding sites (Klinger et al., 2001). Suramin interacted directly with the CaM-binding sites of the ryanodine receptor (RyR) and thereby counteracted the effects of CaM (Papineni et al., 2002). We now report that suramin interacted with the different identified CaM-binding sites on the IP₃R in both the presence and the absence of Ca²⁺. Suramin thereby strongly inhibited IICR. The functional importance of the N-terminal Ca²⁺-independent CaM-binding site in this inhibition was confirmed by the finding that CaM₁₂₃₄, a CaM mutant that is unable to bind Ca²⁺, had the same inhibitory effect on the IP₃R as wild-type CaM. Moreover, other CaM-like Ca²⁺-sensor proteins, such as calcium-binding protein 1 (CaBP1), which was recently shown to strongly inhibit IICR and to interact at the same N-terminal site of the IP₃R (Haynes et al., 2004; Nadif Kasri et al., 2004) inhibited IICR by a mechanism similar to that of CaM. We conclude that suramin and CaM are inhibitors of IP₃ binding by allosteric binding to an N-terminal Ca²⁺-independent site. The same interaction is also responsible for inhibition of IICR, but IICR inhibition only occurs in the presence of Ca²⁺. CaM, however, is not the Ca²⁺ sensor for this inhibition.

	Materials and Methods

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Materials. Suramin was purchased from Sigma Aldrich (Belgium). All other biochemicals were of the highest quality available.

Construction of Plasmids. For the construction of pGEX6p2 vector (Amersham Biosciences, Uppsala, Sweden) encoding aa 1 to 225 of mouse IP₃R1, the coding region was amplified from the pcDNA3.1(+)-mouse IP₃R1 as a template by a PCR reaction with forward-primer 5'-TAACCGGATCCATGTCTGACAAAATGTCGAG-3' and reverse primer 5'-CTCCCGAATTCTCATTTCATGAAAAGCACTATCTTC-3'. The PCR product was digested with BamHI and EcoRI (respective restriction sites are underlined) and cloned into the BamHI/EcoRI-treated pGEX6p2 vector. Constructs were sequenced using the Automated Fluorescent sequencing system (Amersham Biosciences).

Expression of IP₃R. Expression of wild-type IP₃R1 in Sf9 cells using the baculovirus expression system was described previously (Sipma et al., 1999). Wild-type IP₃R1 was permanently expressed in R23–11 triple-IP₃R knockout cells (Sugawara et al., 1997) by electroporation and subsequent selection on 1.2 mg/ml G418 (Geneticin; Invitrogen, Carlsbad, CA) (Miyakawa et al., 1999).

Preparation of Microsomes. Total Sf9 microsomes were prepared as described previously (Yoneshima et al., 1997). R23-11 suspension cells were harvested by centrifugation for 5 min at 4000g and washed twice with phosphate-buffered saline without Ca²⁺ and Mg²⁺. Cell pellets were resuspended in homogenization buffer (10 mM Tris-HCl, pH 7.4, 1 mM EGTA, 0.8 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, and 0.5 µg/ml pepstatin A) and homogenized on ice with a probe sonicator (three times 20 s) (MSE Ltd., Crawley, Surrey, UK). Total microsomes were obtained by centrifugation for 25 min at 125,000g. The membranes were resuspended in isotonic medium (20 mM Tris-HCl, pH 7.4, 300 mM sucrose, 0.8 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride). Microsomal preparations were stored at –80°C.

Purification of GST-Fusion Proteins. For preparation of GST-fusion proteins, pGEX6p2 vector containing the coding sequence for aa 1 to 225 N-terminal of mouse IP₃R1 was transformed into BL21(DE3) Escherichia coli. Colonies were grown overnight in 50 ml of Luria-Bertani medium at 37°C. Luria-Bertani medium (400 ml) was added to this preculture, and bacteria were further grown at 37°C until A₆₀₀ amounted to 0.8. Protein expression was induced by adding 0.1 mM isopropyl-thio--D-galactopyranoside to the bacterial culture, which was further grown at 24°C for another 3 h. Bacterial cells were harvested and lysed by sonication. The soluble fraction was incubated for 2 h with glutathione-Sepharose 4B beads (Amersham Biosciences). After washing the beads, immobilized GST-fusion protein was treated for 2 h at 4°C with PreScission Protease (40 U) (Amersham Biosciences) in cleavage buffer (50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, and 1 mM 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane). After centrifugation of the beads, purified 1 to 225 protein was recovered in the supernatant. Purified protein was dialyzed overnight against phosphate-buffered saline, using Slide-A-Lyzer with a cut-off of 10 kDa (Pierce Biotechnology Inc., Rockford, IL) and stored at –80°C.

Expression and Purification of Recombinant Proteins. CaM, CaM₁₂₃₄, and CaBP1 were expressed and purified as described previously (Nadif Kasri et al., 2003).

CaM-Sepharose Pull-Down Assay. CaM-Sepharose 4B (50 µl; Amersham Biosciences) or Sepharose 4B (control) was incubated with 500 ng of purified IP₃R (Maes et al., 2001) or 10 µg of purified bacterial protein (N-terminal aa 1 to 225 of IP₃R1) for 2 h at 4°C in incubation buffer [i.e., one part Tris-buffered saline (20 mM Tris-HCl, pH 7.2, and 150 mM NaCl) mixed with one part bacterial lysis buffer (Pierce), supplemented with 1 mM -mercaptoethanol]. Unbound protein was removed by washing the Sepharose beads four times with 500 µl of the incubation buffer. Bound IP₃R1 or 1 to 225 protein was eluted by incubating the beads with LDS (Invitrogen) for 10 min at 70°C, and the beads were removed by centrifugation at 20,000g for 1 min. All samples were separated on NuPAGE^® 4–12% Bis-Tris SDS-PAGE gels and analyzed by Western blotting, using a polyclonal antibody directed against a conserved N-terminal region (aa 127–141) of the IP₃R [Rbt475 (1/2500)] as the primary antibody.

Nondenaturing Gel Electrophoresis. The electrophoretic mobility of CaM was evaluated by nondenaturing discontinuous PAGE as described by Laemmli (1970). Nondenaturing gels were run at 25 mA and 4°C under high Ca²⁺ conditions (200 µM free Ca²⁺ in all gel buffers) or low Ca²⁺ conditions (1 mM EGTA in all gel buffers). Quantification of the intensity of the CaM bands was done by Imagequant 4.2 (volume quantitation analyses of the pixel intensities within selected objects; Amersham Biosciences).

[³H]IP₃ Binding. Binding studies were performed as described previously (Sipma et al., 1999). [³H]IP₃ binding was performed at 0°C in 100 µl of binding buffer containing 50 mM Tris/HCl, pH 7.0, 1 mM EGTA, and 10 mM -mercaptoethanol. After 30 min of incubation, 10 µl of -globulin (20 mg/ml) and 110 µl of 20% (w/v) polyethylene glycol in IP₃-binding solution were added, and the samples were rapidly filtered through glass-fiber filters. The amount of purified protein used ranged between 2.5 and 8 µg. Nonspecific binding was determined in the presence of 12.5 µM unlabeled IP₃. Statistical analysis was performed using the paired Student's t test. Values were considered significantly different when P < 0.01.

⁴⁵Ca²⁺ Fluxes. ⁴⁵Ca²⁺ fluxes were performed on saponin-permeabilized Lvec and L15 cells (Miyawaki et al., 1990; Mackrill et al., 1996) in essentially the same way as described previously for A7r5 cells (Missiaen et al., 1999). L15 cells were obtained by stable exogenous expression of IP₃R1 in L cells, whereas Lvec cells represent the control cells expressing the empty plasmid vector (Miyawaki et al., 1990; Mackrill et al., 1996). The cells were seeded in 12-well clusters (Costar, MA) at a density of approximately 4 x 10⁴ cm^–2. Experiments were carried out on confluent monolayers of cells (3 x 10⁵ cells/well) between the 7th and 9th day after plating. Cells were permeabilized by incubating them for 10 min with a solution containing 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 2 mM MgCl₂, 1 mM ATP, 1 mM EGTA, and 20 µg/ml saponin at 25°C. The nonmitochondrial Ca²⁺ stores were loaded for 45 min at 25°C in 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 5 mM MgCl₂, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN₃, and 150 nM free ⁴⁵Ca²⁺ (23 µCi/ml). The cells were then washed twice with 1 ml of efflux medium containing 120 mM KCl, 30 mM imidazole-HCl, pH 6.8, 1 mM EGTA, and 10 µM thapsigargin. Thapsigargin was added to block the endoplasmic reticulum Ca²⁺ pumps. The efflux medium was replaced every 2 min for 18 min, and the efflux was performed at 25°C. Free [Ca²⁺] was calculated by the Cabuf program (ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip) and based on the stability constants given by Fabiato and Fabiato (1979). At the end of the experiment, the ⁴⁵Ca²⁺ remaining in the stores was released by incubation with 1 ml of a 2% SDS solution for 30 min. Ca²⁺ release is plotted as the fractional loss (i.e., the amount of Ca²⁺ released in 2 min divided by the total store Ca²⁺ content at that time). The latter value was calculated by summing in retrograde order the amount of tracer remaining in the cells at the end of the efflux and the amounts of tracer collected during the successive time intervals. The Ca²⁺ release was normalized to the total releasable fraction by 5 µM A23187 [GenBank] , which was taken as 100%.

	Results

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Suramin Interacts with the CaM-Binding Sites on the IP₃R. Three CaM-binding sites have been described on the IP₃R1: a low-affinity site near the N terminus of the IP₃R1 that essentially binds CaM in a Ca²⁺-independent way (Adkins et al., 2000; Sienaert et al., 2002); a high-affinity site in the regulatory domain that mainly binds Ca²⁺/CaM (Yamada et al., 1995); and a third site that only appears after splicing out of S2 (i.e., in peripheral tissues) (Islam et al., 1996; Lin et al., 2000). To determine whether suramin interacts with the CaM-binding sites on the IP₃R1, purified IP₃R1 was pulled down on a CaM-Sepharose column. Figure 1A shows the interaction of CaM with IP₃R1 both in EGTA and in the presence of 200 µM Ca²⁺. The IP₃R binding on the CaM-Sepharose column seemed to be largely Ca²⁺-dependent. This is also compatible with previous findings that a single mutation (W1576A) largely abolished the Ca²⁺ dependent binding to the CaM-Sepharose column (Yamada et al., 1995). We observed, however, that there remained a lower but significant CaM binding in the absence of Ca²⁺ (Fig. 1A, lane 3). This could be a result of either a contribution from the N-terminal CaM-binding site or a partially Ca²⁺-independent interaction with the CaM-binding site in the regulatory domain. The presence of 1 µM suramin during the binding reaction completely abolished the interaction of CaM with the IP₃R1. This inhibition was observed for the interaction in the presence of Ca²⁺ as well as for the much weaker interaction in the absence of Ca²⁺ (Fig. 1A).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect of suramin on the interaction of CaM with the IP3R1. A, immunoblot showing the effects of 1 µM suramin on the interaction of full-size IP3R1 from Sf9 cells with CaM-Sepharose in the absence (1 mM EGTA) and presence of Ca2+ (200 µM Ca2+). B, immunoblot showing the effects of 1 µM suramin on the interaction of recombinantly expressed N-terminal aa 1 to 225 of IP3R1 with CaM in the absence (1 mM EGTA) and presence of Ca2+ (200 µM Ca2+).

To investigate the interaction of suramin with the N-terminal CaM-binding site, we performed pull-down experiments of the recombinant 1 to 225 N-terminal domain on a CaM-Sepharose column. The aa 1 to 225 protein was recombinantly expressed in bacteria. This construct contained the low-affinity CaM-bin ding sites (aa 49–81 and aa 106–128) described previously (Sienaert et al., 2002). Figure 1B shows the interaction of the aa 1 to 225 protein with the CaM-Sepharose column. Suramin suppressed binding to the CaM-Sepharose column in the presence and absence of Ca²⁺. Our results indicate that suramin interacts with the N-terminal CaM-binding site as well as with the CaM-binding site in the regulatory domain.

Suramin Binds to CaM-Binding Peptides of IP₃R1. To map the interaction site of suramin more precisely, we examined the ability of suramin to interact with synthetic peptides representing the CaM-binding domains of the IP₃R1 at both high and low [Ca²⁺].

Using band-shift assays, we have shown previously that CaM interacted with two peptides representing positions in the N terminus of IP₃R1: P49–81 and P106–128 (Sienaert et al., 2002). The interaction was Ca²⁺-independent in that it occurred in both the absence and the presence of Ca²⁺. A representative nondenaturing gel illustrating the interaction of the two peptides with CaM is shown in Fig. 2, A and B. The peptide-CaM complexes migrated as higher molecular weight bands than CaM, whereas the peptides alone do not enter the gel because they are positively charged. However, increasing the suramin concentrations disrupted the interaction between CaM and the peptides. This could be observed by the reappearance of the CaM band as a function of added suramin concentration (Fig. 2, A and B). Quantification of the intensity of the CaM bands in the presence of increasing concentrations of suramin in EGTA or in 200 µM free Ca²⁺ is shown in Fig. 2, C and D. For both peptides and in both conditions (EGTA or Ca²⁺), suramin reversed the interaction with CaM.

View larger version (39K): [in this window] [in a new window]   Fig. 2. The ability of suramin to inhibit CaM binding to the N-terminal CaM-binding site of IP3R1. The effects of suramin on the interaction between CaM and IP3R1 P49–81 (A) and P106–128 (B) were assessed by nondenaturing gel electrophoresis. Peptides (30 µM) and CaM (1.5 µM) were incubated in the absence (1 mM EGTA, top) or in the presence of Ca2+ (200 µM free Ca2+, bottom) and in the presence of increasing concentrations of suramin, followed by electrophoresis and Sypro Orange staining. A and B, the arrow indicates the uncomplexed CaM band. Lane a, CaM alone; lane b, no suramin; lane c, 12.5 µM suramin; lane d, 25 µM suramin; lane e, 37.5 µM suramin; lane f, 50 µM suramin; lane g, 75 µM suramin; lane h, 100 µM suramin. C and D, densitometric analysis of uncomplexed CaM band in the presence of P49–81 (C) or P106–128 (D) and increasing concentrations of suramin, in the absence () or presence of Ca2+ ().

It was originally shown that CaM interacted with aa 1564–1585 in the regulatory domain of IP₃R1 in a Ca²⁺-dependent way (Yamada et al., 1995). Recent data suggested that apoCaM also binds to this region of the IP₃R1 (Adkins et al., 2000). One explanation for this finding is that both forms of CaM bind to closely located or overlapping sites within this region. To test this possibility, we synthesized a series of peptides (Fig. 3A) matching sequences around the cytosolic "1564–1585" site. We extended the peptides N- and C-terminal of the 1564–1585 region. The peptides all contained some similarity with known CaM-binding motifs [IQ or Baa motifs (Rhoads and Friedberg, 1997; Jurado et al., 1999)] (Fig. 3A, Table 1). A representative nondenaturing gel of CaM in the presence of four different peptides in either 200 µM free Ca²⁺ (Fig. 3B) or 1 mM EGTA (Fig. 3C) is shown. In Ca²⁺-containing medium, CaM bound to peptides P1554–1585, P1564–1585, and P1564–1595, whereas in 1 mM EGTA, CaM bound only to P1554–1585. Densitometric analysis of CaM (1.5 µM) bands by ImageQuant 4.2 in the presence of 30 µM peptide is summarized for three independent experiments in Fig. 3D. These band-shift data were also confirmed by measuring fluorescence changes of dansylated CaM (dCaM). dCaM binding to a CaM-binding peptide is known to induce an increase in the emission spectrum of the dansyl moiety. We observed these spectral changes when P1554–1585, P1564–1585, or P1564–1595 was added to a buffer containing dCaM (40 nM) in the presence of Ca²⁺. In the absence of Ca²⁺, spectral changes were observed only when P1554–1585 was added (data not shown). The apparent affinities of CaM binding to the peptides are shown in Table 1. In the presence of Ca²⁺ P1564–1595 shows the highest affinity (K_d, 64 ± 5 nM), whereas in the absence of Ca²⁺, P1554–1585 was the only peptide that bound to CaM. P1554–1573 did not bind CaM in either of the two assays.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Detailed analysis of the CaM-binding site in the regulatory domain of IP3R1. A, schematic representation of the peptides used for the band-shift experiments. Known CaM-binding motifs are indicated. White bars represent IQ domains, and black bars represent Baa motifs. B and C, representative 15% nondenaturing gel of 1.5 µM CaM in the presence of each of the IP3R1 peptides (30 µM): in a 50 mM Tris buffer, pH 7.4, containing either 200 µM free Ca2+ (B) or 1 mM EGTA (C) stained with Sypro Orange; the first lane represents the CaM input. Binding to CaM resulted in complexes with those peptides that did not enter the gel and therefore diminished the intensity of the CaM band, because of the formation of CaM-peptide complexes. D, densitometric analysis of the uncomplexed CaM band in the presence of the IP3R1 peptides. The vertical axis denotes the intensity loss of the CaM bands after interaction with the peptides compared with the value for CaM in the absence of peptide.

The results of the band-shift assays and dCaM assays indicate that the total region investigated here (aa 1554–1595) contains sites with affinities for both Ca²⁺/CaM (K_d, 308 ± 18 nM) and apoCaM (K_d, 508 ± 24 nM). The previously determined boundaries of the cytosolic Ca²⁺/CaM-binding site (Yamada et al., 1995), here represented by P1564–1585, therefore did not contain the apoCaM-binding site.

The effect of increasing concentrations of suramin on the interaction between CaM and P1554–1585 is shown in a representative nondenaturing gel of CaM (Fig. 4A). A densitometric analysis of this interaction is presented in Fig. 4B, showing that suramin interacted directly with P1554–1585 both in the presence and in the absence of Ca²⁺. These data indicate that suramin interferes with all known CaM-binding sites on the IP₃R.

View larger version (19K): [in this window] [in a new window]   Fig. 4. The ability of suramin to inhibit CaM binding to the CaM-binding site of IP3R1 in the regulatory domain. A, representative 15% nondenaturing gel of 1.5 µM CaM in the presence of the IP R1 P1554–1585 (30 µM) and increasing concentrations of suramin in a 50 3mM Tris buffer, pH 7.4, containing either 1 mM EGTA (top) or 200 µM free Ca2+ (bottom) stained with Sypro Orange. Lane a, CaM input; lane b, no suramin; lane c, 12.5 µM suramin; lane d, 25 µM suramin; lane e, 50 µM suramin; lane f, 75 µM suramin; lane g, 100 µM suramin. B, densitometric analysis of uncomplexed CaM band in the presence of P1554–1585 and increasing concentrations of suramin in the absence () or presence of Ca2+ ().

Suramin Inhibits IICR. We assessed the ability of suramin to act as a competitive antagonist to Ca²⁺/CaM and apoCaM modulation of IP₃R1. Ca²⁺ /CaM was found to inhibit IP₃R channel activity and was proposed to be the Ca²⁺ sensor that confers Ca²⁺ inhibition of IP₃R channel activity (Michikawa et al., 1999). We reasoned, therefore, that suramin, by competing for endogenously bound CaM to the IP₃R1, could counteract the effects of CaM, as was demonstrated for the RyR (Papineni et al., 2002). IP₃-induced Ca²⁺ release (IICR) was measured in the presence and absence of suramin in permeabilized L15 and Lvec cells. Western blots indicated a 3/1 ratio for IP₃R1/IP₃R3 for L15 and the reverse ratio for Lvec cells (data not shown). This comparison was made because one major difference between both IP₃R isoforms is that IP₃R3 lacks the Ca²⁺/CaM-binding site in the regulatory domain (Yamada et al., 1995).

In permeabilized L15 or Lvec cells, the addition of IP₃ to the efflux medium induced Ca²⁺ release from the nonmitochondrial internal stores. When suramin was added together with IP₃, IICR was inhibited in a concentration-dependent way. This inhibition occurred to the same extent in both cell lines. Dose response curves for suramin inhibition are presented in Fig. 5A. With 200 nM IP₃ in L15 cells or 1 µM IP₃ in Lvec cells, suramin half-maximally inhibited IICR at a concentration of 30 µM. This concentration is similar to that used for interaction with the CaM-binding site on the RyR (Papineni et al., 2002). For both cell lines, suramin decreased the apparent IP₃ sensitivity (Fig. 5B). In L15 cells and Lvec cells, 100 µM suramin increased the EC₅₀ for IICR from 180 ± 16 nM to 1.09 ± 0.03 µM and from 890 ± 27 nM to 2.1 ± 0.04 µM, respectively. Furthermore, suramin had the same potency at different [Ca²⁺]. This means that the bell-shaped regulation of the IP₃R was maintained (Fig. 5C).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of suramin on IICR. A, Ca2+ release from permeabilized cells, loaded with 45Ca2+ during 45 min, was measured after stimulation with 200 nM (L15 cells, ) or 1 µM (Lvec, ) IP3. Ca2+ release was plotted as a function of increasing concentration of suramin. B, Ca2+ release in L15 and Lvec cells was measured as a function of IP in the absence () or presence () of 100 µM suramin. C, Ca2+ release 3from permeabilized L15 cells, loaded with 45Ca2+ during 45 min, was measured after inducing the cells with 200 nM IP3 at different free [Ca2+] in the presence () or absence () of 100 µM suramin. Ca2+ release was always plotted as the percentage of the maximal releasable fraction by 5 µM A23187 [GenBank] . Results represent the means ± S.E. of three independent experiments each performed twice.

Because suramin was previously shown to interfere with ATP-binding sites of the RyR (Emmick et al., 1994), we also investigated the possibility that suramin might inhibit IICR by interacting with the ATP-binding sites on the IP₃R. Therefore, IICR was measured in permeabilized L15 cells in the presence or absence of 1 mM ATP. As described previously, 1 mM ATP stimulated IICR (Maes et al., 2000, 2001). Suramin however inhibited IICR with the same potency under both conditions, suggesting that its effect was not caused by an interaction with the ATP-binding sites on the IP₃R1 (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6. ATP dependence of the inhibitory effects of suramin on IICR. Ca2+ release from permeabilized L15 cells, loaded with 45Ca2+ during 45 min, was measured after stimulating the cells with 200 nM IP3. Ca2+ release was plotted as a function of increasing concentration of suramin in the presence () or absence () of 1 mM ATP. Ca2+ release was always plotted as the percentage of the maximal releasable fraction by 5 µM A23187 [GenBank] . Results represent the means ± S.E. of three independent experiments, each performed twice.

Suramin Inhibits IP₃ Binding. Because the inhibition by suramin was observed in cells predominantly expressing either IP₃R1 or IP₃R3, we investigated further the possibility that the CaM-binding site in the N-terminal part of the receptor, which is conserved among all isoforms (Vanlingen et al., 2000; Sienaert et al., 2002), could be responsible for this inhibition. CaM was previously shown to strongly inhibit IP₃ binding in a Ca²⁺-independent way (Patel et al., 1997; Sipma et al., 1999; Vanlingen et al., 2000; Sienaert et al., 2002). IP₃ binding was measured on a recombinantly expressed protein containing the N-terminal aa 1 to 581 of IP₃R1 (Lbs1). Suramin (10 µM) inhibited IP₃ binding to Lbs1 by 42 ± 6% (Fig. 7A). Furthermore, we have used a deletion mutant Lbs1 1–225, lacking the N-terminal aa 1 to 225. Lbs1 1–225 was previously shown to bind IP₃ but to completely lack the CaM dependence (Sienaert et al., 2002). Similar to the CaM effects, the effect of suramin was totally abolished on Lbs1 1–225 (Fig. 7A). These data suggest that the inhibition of IP₃ binding by suramin is caused by an allosteric interaction with the N-terminal CaM-binding sites. Scatchard analysis performed in the absence or presence of suramin (10 µM) yielded K_d values of 17 ± 3 nM and 55 ± 4 nM, respectively, whereas B_max values were not significantly different (481 ± 19 and 472 ± 23 pmol/mg; Fig. 7B). This indicates that suramin reduced the affinity of the IP₃-binding site but did not affect the number of IP₃-binding sites. Essentially the same results were obtained for microsomes of R23–11 cells expressing full-size IP₃R1 (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of suramin on [3H]IP3 binding to Lbs1 and Lbs1 1–225. A, [3H]IP3 binding to purified Lbs1 () and Lbs1 1–225 () in presence of indicated concentrations of suramin was expressed as the percentage of the binding measured without suramin. Binding was measured at pH 7.0 in the presence of 1 mM EGTA and 3.5 nM [3H]IP3. B, a Scatchard analysis of [3H]IP3 binding to Lbs1 in the absence or presence of 10 µM suramin is presented. Affinity-purified Lbs1 (1.25 µg) was incubated with 3.5 nM [3H]IP3 at pH 7.0 and increasing concentrations of unlabeled IP3 in the absence () or presence () of 10 µM suramin. Data are expressed as the means ± S.E.M. of at least three experiments.

CaM₁₂₃₄ Inhibits IICR. The inhibitory effects of CaM on IICR were observed only in the presence of Ca²⁺ (Adkins et al., 2000; Michikawa et al., 1999; Missiaen et al., 1999). These data therefore suggested that the Ca²⁺-dependent CaM-binding site in the regulatory domain was responsible for this effect and that CaM could be the Ca²⁺ sensor for the IP₃R. However, mutational studies have clearly shown that mutation of this Ca²⁺-dependent CaM-binding site does not alter the inhibition of IICR by Ca²⁺ (Zhang and Joseph, 2001; Nosyreva et al., 2002). Together with the fact that the CaM-binding site in the regulatory domain is not conserved among all isoforms, these results rather suggest a role for the Ca²⁺-independent CaM-binding site in the N terminus. To clarify the molecular mechanism of the inhibition by CaM, we used CaM₁₂₃₄, a mutant CaM that is unable to bind Ca²⁺. We measured IICR in a ⁴⁵Ca²⁺-flux assay at different free [Ca²⁺] (Fig. 8). In permeabilized L15 cells, the addition of 200 nM IP₃ to the efflux medium induced a Ca²⁺ release from non-mitochondrial internal stores. When 10 µM CaM or CaM₁₂₃₄ was added during the efflux, a similar inhibition in IICR was observed. Not only did CaM₁₂₃₄ have the same potency as CaM, but both forms of CaM were only effective at a minimum free [Ca²⁺] of 300 nM (Fig. 8). Recently, evidence was provided that CaBP1, a member of the neuronal Ca²⁺-sensor family, interacted with the N-terminal CaM-binding site of the IP₃R and mimicked the functional effects of CaM. Adding 10 µM CaBP1 during the efflux resulted in a Ca²⁺-dependent inhibition of IICR very similar to that found for CaM and CaM₁₂₃₄ (Fig. 8). The same results were also observed with Lvec cells (data not shown). The inhibition by CaM₁₂₃₄ clearly shows that CaM does not act as the Ca²⁺ sensor for the inhibition of the IP₃R and that the inhibition of IICR is mediated by the N-terminal Ca²⁺-independent CaM-binding site.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effects of CaM, CaM1234, and CaBP1 on IICR in permeabilized L15 cells. Ca2+ release from permeabilized L15 cells, loaded with 45Ca2+ during 45 min, was measured after stimulating the cells with 200 nM IP3. Ca2+ release was plotted as a function of the free [Ca2+] (). The effects of CaM (), CaM1234 (), and CaBP1 () were measured by adding 10 µM concentrations of CaM, CaM1234, or CaBP1, respectively, during the IP3-induced Ca2+ release. Ca2+ release was always plotted as the percentage of the maximal releasable fraction by 5 µM A23187 [GenBank] . Results represent the means ± S.E. of three independent experiments each performed twice.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Suramin is a polysulfonated napthylurea, which was reported to counteract CaM effects by binding to the CaM-binding sites of the RyR (Papineni et al., 2002). Our results indicate that suramin interacts in a competitive manner with all previously identified CaM-binding sites on the IP₃R. This interaction was seen in the presence and the absence of Ca²⁺ (Figs. 2 and 4).

We expected that suramin would counteract the effects of CaM and thereby stimulate IICR, as was previously demonstrated for the RyR. Surprisingly, however, suramin inhibited IICR in ⁴⁵Ca²⁺ flux assays (Fig. 5). As suramin inhibited IICR for IP₃R1 as well as for IP₃R3, we reasoned that the functional effects of suramin could be caused by its interaction with the conserved N-terminal CaM-binding domain. The absence of the suramin effects on IP₃ binding to a mutant lacking the N-terminal CaM-binding sites (1–225) clearly showed that suramin allosterically inhibited IP₃ binding (Fig. 7). Effects on IICR can therefore be explained as a decrease in the apparent IP₃ affinity.

These data are at variance with previous models regarding the regulation of the IP₃R by CaM. It has been proposed that CaM is responsible for the Ca²⁺-dependent inactivation of the IP₃R. The only known Ca²⁺-dependent CaM-binding site, however, is not conserved in all three isoforms. Moreover, two studies have shown that mutation of the high-affinity CaM-binding site in the regulatory domain altered neither the Ca²⁺ dependence of the IP₃R nor the effects of CaM (Zhang and Joseph, 2001; Nosyreva et al., 2002). Based on the latter findings, it has been suggested that the N-terminal Ca²⁺-independent CaM-binding site could be responsible for inhibition of IICR that was observed for the three isoforms (Adkins et al., 2000; Zhang and Joseph, 2001; Nosyreva et al., 2002). However, the Ca²⁺-dependent effect of CaM could not be correlated with the Ca²⁺-independent binding of CaM to that site. Therefore, we have used CaM₁₂₃₄, a mutant CaM that is unable to bind Ca²⁺. CaM₁₂₃₄ inhibited IICR in a Ca²⁺-dependent way with the same potency as CaM. At low [Ca²⁺], CaM₁₂₃₄ had no effect on IICR; at higher [Ca²⁺], CaM₁₂₃₄ inhibited IICR. These data clearly show that there is a Ca²⁺-dependence for the action of CaM, but CaM is not the Ca²⁺ sensor for this Ca²⁺ dependence. Electron microscopy of the IP₃R clearly demonstrated that the conformation of the IP₃R dramatically changes in the presence of Ca²⁺ from a closed to an open "windmill" structure (Hamada et al., 2002, 2003). It is conceivable that CaM tethered to the IP₃R may exert its inhibitory role on the IP₃R only in the open "windmill" configuration.

The functional significance of the CaM-binding site in the regulatory domain still remains an open question. We found that this CaM-binding site seems to be more complex than was described previously (Yamada et al., 1995) and consists of a high-affinity Ca²⁺/CaM and a lower-affinity apoCaM-binding site (Fig. 3). We can exclude, based on work by Zhang and Joseph (2001) and Nosyreva et al. (2002), the possibility that this site is responsible for the Ca²⁺-dependent inhibition of the IP₃R. We cannot, however, exclude the possibility that this CaM-binding site indirectly regulates the IP₃R because previous data have shown that CaM binding may depend on the phosphorylation status of the IP₃R1 (Lin et al., 2000).

On the other hand, the lower-affinity CaM-binding site on the N terminus seems to be involved in the direct regulation of the IP₃R by affecting the sensitivity to IP₃. Our finding that suramin mimicked the inhibitory effect of CaM suggests that the N-terminal CaM-binding site may be a protein-protein interaction site involved in structural intramolecular associations, similar to that proposed for RyRs (Slavik et al., 1997; Sencer et al., 2001). The inhibitory effect of both suramin and CaM may then result from a disruption of such an intramolecular interaction. The Ca²⁺ dependence of the inhibition would suggest that this intramolecular interaction is stabilized in the "windmill" configuration of the IP₃R. We cannot exclude, however, the possibility that suramin may inhibit IICR by removing endogenously bound CaM from an as-yet unidentified high-affinity site (Lin et al., 2000). Other members of the CaM family, such as Ca²⁺-binding proteins (CaBPs), were also shown to interact with the N-terminal part of all IP₃Rs and thereby inhibited IP₃R function (Haynes et al., 2004; Nadif Kasri et al., 2004). There was evidence that CaBP1 interacted at the same binding site as CaM and CaM₁₂₃₄ (Nadif Kasri et al., 2004). In another report, however, it was claimed that CaBP1 was an activator of the IP₃R by interaction with the N-terminal part of the IP₃R (Yang et al., 2002). From the present data, we conclude that CaBP1 inhibited IICR in essentially the same way as CaM or CaM₁₂₃₄.

Taken together, our data show that suramin is a potent inhibitor of the IP₃R. It mimicked the effect of CaM, CaM₁₂₃₄, and CaBP1 by inducing an allosteric but competitive inhibition of IP₃-binding. The inhibition of IP₃ binding via the N-terminal CaM-binding site also resulted in lower IP₃ sensitivity for IICR. However, CaM and CaM₁₂₃₄ only inhibited IICR in the presence of Ca²⁺, which indicates that a Ca²⁺-dependent conformational change is required, but CaM is not the Ca²⁺ sensor for this change.

Further mutational analysis of the N-terminal CaM-binding site should reveal its potential role in the intramolecular interactions required for linking IP₃ binding to IICR.

	Acknowledgements

 
We thank Lea Bauwens, Marina Crabbé, Silvie De Swaef, and Silvia Vangeel for their technical assistance. The mammalian CaM cDNA was kindly provided by Dr. Z. Grabarek (Boston, MA) and the rat cDNA for CaM₁₂₃₄ was kindly provided by Dr. J. Adelman (Portland, OR).

	Footnotes

 
Work performed by the authors was supported by grants G.0210.03 (to H.D.S. and J.B.P.) and G.O206.01 (to L.M.) of the Fund for Scientific Research Flanders (Belgium), and by grant 99/08 from the Concerted Actions of the Katholieke Universiteit Leuven (to L.M., H.D.S., G.C., and J.B.P.). G.B. is a postdoctoral fellow of the Fund for Scientific Research Flanders (Belgium) and has a fellowship of the Belgian American Educational Foundation (B.A.E.F.). R.A.F. has a senior postdoctoral fellowship of the Fund for Scientific Research Flanders (Belgium). We are also grateful for the support by the Interuniversity Poles of Attraction Programme-Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs, IUAP P5/05.

ABBREVIATIONS: IP₃R, inositol 1,4,5-trisphosphate receptor; IP₃, inositol 1,4,5-trisphosphate; CaM, calmodulin; [Ca²⁺], calcium concentration; RyR, ryanodine receptor; IICR, IP₃-induced Ca²⁺ release; aa, amino acid(s); apoCaM, apocalmodulin; dCaM, dansylated CaM; CaBP, calcium-binding protein; A23187 [GenBank] , 4-bromocalcimycin.

Address correspondence to: Humbert De Smedt, Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000, Leuven, Belgium. E-mail: humbert.desmedt{at}med.kuleuven.ac.be

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