Modulation of Inositol 1,4,5-Trisphosphate Binding to the Recombinant Ligand-binding Site of the Type-1 Inositol 1,4,5-Trisphosphate Receptor by Ca2+ and Calmodulin*

Henk SipmaDagger , Patrick De Smet§, Ilse Sienaert§, Sara Vanlingen, Ludwig Missiaen, Jan B. Parys, and Humbert De Smedtparallel

From the Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium

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A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 ± 15 nM in the absence to 196 ± 18 nM in the presence of 5 µM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 µM Ca2+, 42.0 ± 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 ± 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 µM. Maximal inhibition (10 µM calmodulin, 43.1 ± 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 ± 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 µM Ca2+ (together 74.5 ± 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.

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Most cell types express inositol 1,4,5-trisphosphate receptors (IP3Rs)1 mediating IP3-induced release of Ca2+ from internal Ca2+ stores. Three IP3R isoforms that differ in structure, IP3 binding characteristics, and regulation have been identified (1).

Submicromolar [Ca2+] inhibits IP3 binding to cerebellar microsomes (2-7) and to microsomes of Sf9 insect cells expressing IP3R-1 (8, 9). Inhibition of IP3 binding to IP3R-1 by Ca2+ might constitute one of the components inducing the descending phase of the bell-shaped dependence of IP3-induced Ca2+ release on cytoplasmic Ca2+ (10-13). There is still controversy about the molecular mechanism responsible for the inhibitory effect of Ca2+ on IP3 binding, because experiments on purified IP3R-1 have given conflicting results. It was suggested that Ca2+ acts directly on a Ca2+-binding site on IP3R-1 (7). On the other hand, indirect inhibition via an accessory Ca2+-binding protein like calmedin was also reported (4). Moreover, it is still a matter of debate whether Ca2+-induced inhibition is caused by a decrease in the affinity of IP3R-1 for IP3 (5, 7, 8) or by a Ca2+-induced reduction in IP3-binding sites (9).

Recently, calmodulin was reported to cause inhibition of IP3 binding (14, 15). Calmodulin would in this case bind to a site different from the Ca2+-dependent calmodulin-binding site found earlier (16), because calmodulin was able to bind to IP3R-1 and inhibit IP3 binding to IP3R-1 in a Ca2+-independent manner (14, 15).

The N-terminal part of the IP3R contains all the structural determinants responsible for specific and selective binding of its physiological agonist, IP3 (17-20). We have therefore expressed the N-terminal ligand-binding site (first 581 amino acids) of the mouse IP3R-1 in Escherichia coli, using a strategy of growth and expression at low temperatures, as described previously by Yoshikawa et al. (20). This protein contains a previously identified Ca2+-binding region located between amino acids 304-450 (21). We now demonstrate that Ca2+ and calmodulin can both inhibit IP3 binding to this recombinant protein and that these inhibitors act independently and additively. Our data indicate that the N-terminal ligand-binding domain of IP3R-1 contains regulatory regions directly interacting with Ca2+ and calmodulin.

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Expression of IP3R-1 in Sf9 Insect Cells-- The full-length neuronal mouse IP3R-1 cDNA clone containing the S1 splice domain in p400C1 plasmid vector (22) was kindly provided by Drs. K. Mikoshiba and A. Miyawaki (University of Tokyo, Tokyo, Japan). The 5'-untranslated region of the original p400C1 clone was removed by polymerase chain reaction (PCR) by amplification of the 5'-terminal part up to the Cel-II restriction site (nucleotide 555), before subcloning the IP3R-1 cDNA in the baculovirus (Autographa californica) transfer vector pVL 1393 (Invitrogen). Recombinant virus was produced in Spodoptera frugiperda (Sf9) cells by cotransfection of the pVL 1393 IP3R-1 construct and the linearized A. californica nuclear polyhydrosis virus DNA (BaculoGold, Pharmingen). The recombinant viruses were purified by isolating individual plaques of transfected cells. These clonal viral populations were amplified by infecting Sf9 cells. The recombinant protein was harvested 2 days after infection of the Sf9 cells with the amplified virus at a multiplicity of infection of 2-6.

Construction of a Vector Encoding the Ligand-binding Site of IP3R-1-- A bacterial expression vector (pET 21b/+, Novagen Inc.) was used to express the N-terminal 581 amino acids of the mouse IP3R-1, including the S1 splice site. PCRs were performed to produce two DNA fragments containing the coding sequence for 1) amino acid 1-64 and 2) amino acid 471-581, using SalI cut p400C1 containing cDNA (9448 base pairs) encoding the mouse IP3R-1 (22) as a template. Fragment 1 was produced with forward primer 5'-CGCGCATATGTCTGACAAAATGTCG-3' (containing an NdeI site including the start codon) and reverse primer 5'-CGGAGTATCGATTCATAGG-3' (containing the codons for amino acids 64-65 including a ClaI site). Fragment 2 was synthesized with forward primer 5'-GGTCTGTCACGAAGCTTTTGG-3' (containing a HindIII site) and reverse primer 5'-GTATGCGGCCGCTTACATGAAGCCAAACTGCTTGG-3' (containing the codon for methionine-581, directly followed by a stop codon and a NotI site). PCR fragment 2 was cut with HindIII and NotI and ligated into the HindIII/NotI site of pcDNA3.1+ (Invitrogen), yielding pcDNA-F2. The SalI digest of p400C1 containing a 329-base pair 5' noncoding region and the complete coding region of IP3R-1 was first subcloned in the pCI expression plasmid (Promega) to yield pCI-IP3R-1. A 1751-base pair fragment was obtained from pCI-IP3R-1 by partial NheI/HindIII digest and ligated into the NheI/HindIII sites of the pcDNA-F2 construct. Subsequently, the EcoRI/NotI fragment of pcDNA-F2 was ligated into the EcoR-I/NotI sites of the bacterial expression vector pET 21b/+. To remove the 5' noncoding sequences, the resulting construct and PCR fragment 1 were digested with NdeI/ClaI and ligated, yielding pET-581. The sequences of pET-581 and of the PCR-amplified part of the pVL 1393 IP3R-1 construct were confirmed by double-stranded sequencing using the Automated Laser FluorescentTM sequencing system (Amersham Pharmacia Biotech).

Expression in E. coli-- The expression of the recombinant N-terminal 581 amino acids of the IP3R-1 (Lbs-1 (ligand-binding site-1)) was performed essentially as described by Yoshikawa et al. (20). A single colony of E. coli BL21(DE3) transformed with pET-581 was resuspended in 2 ml of LB medium containing 100 µg/ml ampicillin and grown overnight at 27 °C. 1 ml of this culture was diluted in 50 ml of fresh medium (100 µg/ml ampicillin) and grown at 21 °C for 10 h to an A600 of about 1.5. Subsequently, expression of the recombinant protein was induced in the presence of isopropyl-1-thio-beta -D-galactopyranoside (0.75 mM) for 20 h at 14 °C. Cells were harvested by centrifugation and washed with a buffer containing 10 mM KH2PO4, 30 mM NaHPO4, 153 mM NaCl, pH 7.5.

Preparation of the Soluble Fraction of E. coli-- The cell pellet was resuspended in 5 ml of homogenization buffer (HB) containing 10 mM Tris-HCl, pH 7.4, 1 mM beta -mercaptoethanol, 0.8 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 1 µM pepstatin A, and 75 nM aprotinin. This cell suspension was digested with lysozyme (0.1 mg/ml) for 30 min at 4 °C, followed by six cycles of freeze-thawing (in liquid nitrogen and at 37 °C) and sonication at 12 kHz, two times for 15 s (probe sonicator, MSE Ltd., Westminster, UK). After centrifugation (30,000 × g, 60 min), the supernatant containing the soluble fraction of E. coli was stored at -80 °C.

Partial Purification of the Recombinant Protein-- The soluble fraction containing the recombinant protein (Lbs-1) and supplemented with 0.3 M NaCl was loaded on a heparin-agarose column (80 µl beads/mg protein). Before loading, the column was pre-equilibrated with HB containing 0.3 M NaCl. The column was washed with 5 volumes HB supplemented with 0.3 M NaCl, and Lbs-1 was eluted with 2.5 volumes HB supplemented with 0.8 M NaCl.

IP3 Binding-- [3H]IP3 binding was performed in 100 µl of a solution containing 50 mM Tris-HCl, pH 7.0 or 7.8, 50 mM NaCl, 1 mM EGTA or BAPTA, 10 mM beta -mercaptoethanol, 2.5 µg of partially purified Lbs-1, and variable concentrations of [3H]IP3 (see figure legends) at 0 °C for 30 min. Subsequently, 10 µl of gamma -globulin (20 mg/ml) and 110 µl of 10% polyethylene glycol in IP3 binding buffer was added for 10 min, and the mixture was quickly filtered through glass fiber filters and washed using a Combi Cell Harvester (Skatron). Activity on the filters was quantified with a Beckman scintillation counter. Nonspecific binding was determined in the presence of 10 µM unlabeled IP3. Routinely, specific [3H]IP3 binding amounted to more than 95% of total binding. Scatchard analyses were performed using the Kell Radlig program (version 5.0.4, Biosoft, Cambridge, UK). A Student's t test (paired or unpaired) was used for statistical analyses. Values were considered statistically different when p < 0.05.

Microsomes, Antibodies, and Western Blotting-- Microsomes of rabbit cerebellum and RBL-2H3 cells (F3 fraction) were prepared as described by Parys et al. (23) and Vanlingen et al. (24), respectively. Microsomes of 16HBE14o- cells and Sf9 insect cells were prepared as described by Sienaert et al. (25) and Yoneshima et al. (8), respectively. Full-length IP3R-1 was detected with the polyclonal antibody Rbt03. This antibody is directed against the same epitope as the previously described Rbt04 (23-26) and has specificity and affinity identical to those of Rbt04. A second antibody was raised against the Ca2+-binding domain cyt3b present between amino acids 378 and 450 of the mouse IP3R-1 (21). Two rabbits were injected subcutaneously and intramuscularly with Freund's complete adjuvant containing 0.5 mg of cyt3b fused to glutathione S-transferase. Animals were boosted 2 weeks later with the same antigen in Freund's incomplete adjuvant and regularly thereafter. After three boost injections, both rabbits produced high titers of antibody. Both these antibodies (named anti-cytI3b-1 and anti-cytI3b-2) reacted specifically with IP3R-1 from multiple species including rat, rabbit, and Xenopus. The partially purified soluble fraction of E. coli was analyzed by SDS-PAGE on a 3-12% linear gradient and either stained with Coomassie Blue or SyproTM Orange or transferred to Immobilon-P (Millipore). Blots were blocked for 1 h in a buffer containing 10 mM NaH2PO4, 32 mM Na2HPO4, pH 7.5, 154 mM NaCl, 0.1% Tween 20, and 5% milk powder and incubated with the primary antibody for 1 h in the same buffer without milk powder. Alkaline phosphatase-coupled anti-rabbit antibody was used as secondary antibody. The immunoreactivity was visualized by conversion of the VistraTM ECF substrate into a fluorescent probe (Amersham Pharmacia Biotech) and scanned with the Storm 840 FluorImager, equipped with the Imagequant NT4.2 software (Molecular Dynamics) as described previously (24, 26).

Materials-- Adenophostin-A was a gift of Dr. S. Takahashi (27). IP3 was obtained from Roche Molecular Biochemicals. [3H]IP3 was from Amersham Pharmacia Biotech. Restriction enzymes were from New England Biolabs Inc. or Roche Molecular Biochemicals. T4-DNA ligase was from Life Technologies, Inc. High purity bovine brain calmodulin was from Calbiochem. Polyethylene glycol, gamma -globulins, and heparin-agarose beads were obtained from Sigma . SyproTM Orange was from Bio-Rad. Ethylene glycolbis-(sulfosuccinimidylsuccinate) was from Pierce.

    RESULTS AND DISCUSSION
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Expression of the Full-length Mouse IP3R-1 in Sf9 Insect Cells-- Microsomes of Sf9 insect cells transfected with pVL 1393 IP3R-1 (Sf9-IP3R-1) were immunoreactive to an antibody specifically recognizing a C-terminal epitope of mouse IP3R-1 (Fig. 1A). Under identical conditions, no IP3R was detected in control pVL 1393 transfected Sf9 cells. The expressed IP3R-1 migrated on SDS-PAGE with the same apparent molecular mass as observed for IP3R-1 from rabbit cerebellar microsomes and amounted to 2.5 times the value of the latter (Fig. 1A). In the presence of 5 nM [3H]IP3, microsomes of Sf9 cells expressing IP3R-1 specifically bound IP3. The binding activity (589 ± 83 fmol/mg at pH 7.8) was also about 2.5 times the value found for rabbit cerebellar microsomes (252 ± 43 fmol/mg). Microsomes of control pVL 1393 transfected Sf9 cells showed no significant IP3 binding under these conditions.


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Fig. 1.   Expression of mouse IP3R-1 in Sf9 insect cells and expression of the recombinant IP3-binding domain of IP3R-1 in E. coli. A, microsomes of Sf9 insect cells transfected with pVL 1393-IP3R-1 bearing the complete coding sequence of mouse IP3R-1 (lane 1, 10 µg), Sf9 insect cells transfected with pVL 1393 (lane 2, 10 µg), or rabbit cerebellum microsomes (lane 3, 10 µg) were loaded on a 3-12% SDS-PAGE and blotted to Immobilon-P. IP3R-1 (arrow) was stained with the Rbt03 polyclonal antibody raised against the C-terminal part of mouse IP3R-1. The relevant part of the blot is shown. B, immunoreactivity to the anti-cytI3b-1 polyclonal antibody (dilution 1:500) of microsomes of rabbit cerebellum (lane 1, 10 µg) expressing IP3R-1, RBL-2H3 rat basophilic leukemia cells (lane 2, 50 µg) expressing predominantly IP3R-2 (36), and 16HBE14o- human bronchial epithelial cells (lane 3, 200 µg) expressing predominantly IP3R-3 (25). The complete blot is shown. Only IP3R-1 (arrow) was detected. C, partially purified soluble fraction of E. coli transfected with control pET 21b/+ (lane 1, 50 µg) or with pET-581 (lane 2, 7.5 µg; lane 3, 20 µg; lane 4, 50 µg) was loaded on a SDS-PAGE and stained with Coomassie Blue. Lanes 5-9 represent an immunoblot of the same samples as shown in lanes 1-4: partially purified fraction of E. coli transfected with control pET 21b/+ vector (lane 5, 5 µg) or transfected with the pET-581 vector (lane 6, 0.25 µg; lane 7, 0.75 µg; lane 8, 1.5 µg; lane 9, 5 µg). Anti-cyt3b-1 (1/1250) was used to detect Lbs-1. The arrow indicates the position of the Lbs-1 recombinant protein. The relevant part of the blot is shown, and the molecular masses of the protein standards are indicated. These data are representative for three analyses with nearly identical results.

Expression of the IP3-binding Domain of the IP3R-1-- We constructed a bacterial expression vector containing the coding sequence of the N-terminal 581 amino acids of the mouse IP3R-1 (pET-581). The recombinant protein (Lbs-1) was expressed in E. coli using a strategy described earlier by Yoshikawa et al. (20). Lbs-1 was partially purified on a heparin-agarose column and migrated with an apparent molecular mass of 66 kDa on a SDS-polyacrylamide gel (Fig. 1C). The protein reacted with the anti-cytI3b-1 polyclonal antibody, which specifically recognized an epitope in the N terminus of the IP3R-1 (Fig. 1, B and C). Although some degradation of the 66-kDa protein occurred, it was determined by quantitative analysis of the fluorescence signals obtained on immunoblots (Fig. 1C) and of the proteins separated by SDS-PAGE and stained with the fluorescent dye SyproTM Orange (data not shown) that at least 80% of the recombinant protein was in the intact form. Degraded proteins missing the primary antibody-epitope (amino acids 378-450 (21)) or smaller than 40 kDa will not bind IP3 (20) and will therefore not influence IP3 binding measurements. The isolated IP3-binding domain was likely to exist as a monomer, because it did not contain structural elements necessary for multimerization (18, 28). We have investigated possible multimerization of Lbs-1 by chemical cross-linking experiments. After treatment with the N-hydroxysuccimide ester, ethylene glycolbis-(sulfosuccinimidylsuccinate) (0.25 mM), and separation of proteins with denaturating SDS-PAGE, we could clearly detect dimers of glutathione S-transferase (data not shown), a protein known to form dimers (29, 30) and used here as positive control. However, this treatment failed to covalently link molecules of Lbs-1 with each other or with other proteins in the suspension, either in the presence or the absence of 5 µM IP3, 1 µM adenophostin-A, or 5 µM Ca2+ (data not shown). These results strongly suggest that Lbs-1 is a monomeric polypeptide.

Characterization of IP3 Binding to Lbs-1-- Fractions of partially purified Lbs-1 specifically bound [3H]IP3 at a pH of 7.8, whereas a similar fraction from E. coli that was only transformed with host pET 21b/+ did not bind IP3 under these conditions. [3H]IP3 was displaced from Lbs-1 by unlabeled IP3 with an IC50 of 60 nM (Fig. 2). The displacement data could best be fitted using a single-site Scatchard model, yielding a Kd of 46 ± 4 nM, a Bmax of 280 ± 60 pmol/mg, and a Hill coefficient of 1.1 ± 0.1 (Fig. 2, inset). These values are very close to values found for the intact purified mouse cerebellum IP3R-1, obtained under similar experimental conditions (IC50, 76 nM; Hill coefficient, 1.1 (31); Kd, 37 nM (20)). Furthermore, we have previously reported a Kd of 46 ± 17 nM for the purified Xenopus IP3R-1 under identical experimental conditions but at a slightly higher pH (13). These findings are in agreement with earlier reports, demonstrating that recombinant proteins, containing the first 788 (17), 734 (20), or 576 amino acids (19) of the IP3R-1 showed similar specificity for inositol phosphates and similar affinities for IP3 as the intact IP3R-1. These observations indicate that Lbs-1 is in the right conformation to act as a bona fide IP3-binding pocket.


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Fig. 2.   Binding of IP3 to Lbs-1. Increasing concentrations of IP3 displaced [3H]IP3 from partially purified Lbs-1 (2.5 µg) with an IC50 of 60 nM. [3H]IP3 binding was determined in the presence of 2.5 nM [3H]IP3 at pH 7.8 after rapid filtration of samples through glass fiber filters. Specific binding amounted to 8.7 ± 1.4 pmol/mg protein (typically about 2300 dpm). Data are presented as the means ± S.E. of three experiments consisting of independent triplicates. A Scatchard analysis of a representative set of data is presented in the insert. Scatchard analyses yielded a Kd value of 46 ± 4 nM and a Bmax of 280 ± 60 pmol/mg (n = 3).

Effect of Ca2+ on IP3 Binding to Sf9-IP3R-1 and Lbs-1-- Submicromolar [Ca2+] was found to inhibit IP3 binding to IP3R-1 from cerebellum (2-7) or expressed in insect Sf9 cells (8, 9). Lbs-1 contains two amino acid sequences (304-381 and 378-450) that were found to bind Ca2+ (21). These sites could be involved in the inhibitory effect of Ca2+. In our hands, micromolar Ca2+ (5 µM) inhibited IP3 binding to microsomes of insect Sf9 cells expressing mouse type-1 IP3R by 33.8 ± 10.2% at a physiological pH of 7.0 (Fig. 3A). IP3 binding to Lbs-1, which includes the S1 splice site, was inhibited to the same extent by 5 µM Ca2+ under identical experimental conditions (42.0 ± 6.4%, Fig. 3B). This inhibition was less pronounced than the maximal Ca2+-induced inhibition of IP3 binding observed in microsomes of Sf9-IP3R-1 cells (lacking S1) by Yoneshima et al. (70% (8)) and Cardy et al. (54% (9)). Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 within a physiological range between 30 nM and 5 µM. A half-maximal effect occurred at about 200 nM (Fig. 4A). Scatchard analyses performed in the absence or presence of Ca2+ (5 µM) yielded Kd values of 115 ± 15 and 196 ± 18 nM IP3, respectively, whereas the Bmax values were not significantly different (330 ± 30 and 410 ± 60 pmol/mg, respectively) (Fig. 5). This indicates that Ca2+ reduced the affinity of the IP3-binding site without an effect on the number of binding sites. Furthermore, because Lbs-1 was isolated in the absence of Ca2+ chelators, the protein was exposed to micromolar Ca2+ concentrations for some time. The fact that after this treatment IP3 binding was higher in the presence of only Ca2+ chelators than in the presence of chelators and (sub)micromolar free Ca2+ indicates that the effect of Ca2+ on IP3 binding is reversible. The Ca2+-induced reduction of affinity of Lbs-1 for IP3 is in agreement with the results obtained on rat cerebellar microsomes (5), on microsomes of insect Sf9-IP3R-1 cells (8), and on immunopurified sheep cerebellum IP3R-1 (7). In a study of the intact IP3R-1 expressed in insect cells, it was also suggested that the inhibitory effect of Ca2+ might be due to a reduction in the number of IP3-binding sites (9). Our binding data do not indicate a decrease in binding sites for IP3. The binding data in the absence and presence of Ca2+ are compatible with a one-site model of IP3 binding, in agreement with the monomeric nature of our protein preparation. It should be pointed out, however, that a different observation with respect to the mechanism of the inhibition may be indicative for a more complex role of cytosolic Ca2+ on the intact receptor as compared with the isolated IP3-binding domain. Because there are at least five additional potential interaction sites with Ca2+ in the cytosolic domains (21), a more complex dependence on Ca2+ for the intact receptor is not unexpected. Our data indicate that there is a direct interaction of Ca2+ with the IP3-binding domain, but this interaction may represent only part of the feedback mechanism that controls IP3-induced Ca2+ release.


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Fig. 3.   Binding of IP3 to Lbs-1 and microsomes of Sf9-IP3R-1 insect cells. [3H]IP3 binding to microsomes of Sf9-IP3R-1 cells (100 µg, A) and partially purified Lbs-1 (2.5 µg, B) was measured in the presence or absence of Ca2+ (5 µM) and/or calmodulin (10 µM) and was expressed as the percentage of binding in the absence of these modulators (control). Binding was measured at pH 7.0 in the presence of 1 mM EGTA, 3.6 nM [3H]IP3. Sf9-IP3R-1 microsomes and partially purified Lbs-1 were pre-exposed to Ca2+ and/or calmodulin for 5 min before adding [3H]IP3. Specific binding of IP3 to microsomes of Sf9-IP3R-1 cells amounted to 151 ± 18 fmol/mg (about 1000 dpm), whereas microsomes of pVL 1393-transfected Sf9 cells did not significantly bind IP3. Specific binding to Lbs-1 was 8.5 ± 0.7 pmol/mg (2000 dpm, no binding to soluble fraction of control bacteria treated the same way as the Lbs-1 containing fraction). *, significantly different from binding in the absence of modulators. Data are expressed as the means ± S.E. of at least four experiments, consisting of independent quadriplicates.


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Fig. 4.   The effect of different [Ca2+] on IP3 binding to Lbs-1. [3H]IP3 binding to partially purified Lbs-1 (2.5 µg) in the presence of the indicated concentrations of Ca2+ was expressed as a percentage of the binding measured in Ca2+-free buffer with 1 mM EGTA at pH 7.0 (A) or 1 mM BAPTA at pH 7.8 (B). The different Ca2+ concentrations were buffered with 1 mM EGTA or BAPTA and calculated with the Maxchelator software (Dr. C. Patton, Stanford University, Stanford, CA). *, significantly different from binding in Ca2+-free buffer. Data are expressed as the means ± S.E. of at least four experiments, consisting of independent quadriplicates.


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Fig. 5.   Scatchard analysis of IP3 binding to Lbs-1 in the presence and absence of Ca2+. Partially purified Lbs-1 (3.75 µg) was incubated with 3.6 nM [3H]IP3 at pH 7.0 and increasing concentrations of unlabeled IP3 in the absence (open circle ) or presence () of 5 µM Ca2+. Data are expressed as means of three independent determinations of one experiment repeated twice with similar results.

Remarkably, the effect of Ca2+ on IP3 binding was pH-dependent. No inhibition was observed at a pH of 7.8 (Fig. 4B). In these experiments, BAPTA was used as chelating agent. It has been suggested that high doses of Ca2+-free chelators, especially BAPTA, can inhibit IP3 binding (32, 33). The inhibitory effect of Ca2+ on IP3 binding might therefore be shielded by a stimulatory effect of Ca2+ in relieving inhibition by the chelator. IP3 binding to Lbs-1 (pH 7.8) was, however, identical in the presence of 1 mM BAPTA, 0.1 mM BAPTA, 0.1 mM BAPTA and 5 µM free Ca2+, and 1 mM EGTA (data not shown). Therefore, we can exclude the possibility that the absence of an effect of Ca2+ on IP3 binding at pH 7.8 was caused by effects of BAPTA. As shown above, lowering the pH from 7.8 to 7.0 caused an increase of the Kd value for the binding of IP3 to pET-581 from 46 to 115 nM. The enhancement of IP3 binding to the IP3R-1 at a higher pH is a well documented phenomenon (5, 34). It can be suggested that the different conformational states of the IP3R-1 that apparently accompany changes in pH are unequally susceptible to inhibition by Ca2+.

Because Lbs-1 was expressed in a bacterial environment, the inhibitory effect of Ca2+ on IP3 binding to Lbs-1 strongly suggests direct binding of Ca2+ to the N-terminal IP3-binding domain of IP3R-1 and strongly disfavors the idea of involvement of accessory proteins, such as calmedin (4). The hypothesis of direct binding of Ca2+ to IP3R-1 is in agreement with results obtained by Picard et al. (7), who showed that Ca2+ could still inhibit IP3 binding to rat cerebellar microsomes after removal of peripheral membrane proteins with high alkaline treatment and to immunopurified sheep cerebellar IP3R.

We have previously demonstrated direct Ca2+ binding (21) to a stretch of amino acids (304-450) located in the "core" IP3-binding domain (20). Most likely, Ca2+ exerts its inhibitory effect on IP3 binding by interacting with this particular region. Our data indicate that the modulation of the IP3 affinity by cytosolic Ca2+ is an inherent property of the IP3-binding domain. Unfortunately, it may be very difficult, if not impossible, to determine the amino acid residues critically involved in the inhibitory effect of Ca2+ on IP3 binding because mutations and deletions in this region will almost certainly also affect the characteristics of IP3 binding or eliminate IP3 binding (20).

Our data give further strong support to the idea that Ca2+, in a physiological range, is able to inhibit IP3 binding to IP3R-1. This mechanism is likely to contribute to feedback inhibition of IP3-induced Ca2+ release by (sub)micromolar [Ca2+] (10-13).

Effect of Calmodulin on IP3 Binding to Lbs-1-- In the presence of Ca2+, calmodulin binds to the regulatory domain of the type-1 and -2 IP3R (16). Recently, it was shown that calmodulin can also bind to IP3R-1 in the absence of Ca2+, thereby inhibiting IP3 binding to the purified cerebellar IP3R (14) and to microsomes of insect Sf9 cells expressing rat IP3R-1 (15). In our hands, calmodulin (10 µM) inhibited IP3 binding to microsomes of Sf9 cells expressing IP3R-1 and to Lbs-1 by 35.8 ± 8.7 and 43.1 ± 5.9%, respectively (Fig. 3, A and B). These values were similar to maximal inhibition (10 µM calmodulin) reported for Sf9 microsomes (40% (15)) and rat cerebellum microsomes (36% (14)). In the presence of both Ca2+ (5 µM) and calmodulin (10 µM), IP3 binding was inhibited by 74.5 ± 5.1% (i.e. 50% of the level in the presence of only Ca2+). Therefore, inhibition by calmodulin was Ca2+-independent and was additive to inhibition by Ca2+, as suggested previously for the intact IP3R-1 (14, 15). As was also observed for the effect of Ca2+, inhibition of IP3 binding by calmodulin does not seem to depend on the presence or absence of the S1 splice domain. The effect of calmodulin on IP3 binding to Lbs-1 was concentration-dependent (Fig. 6A). Calmodulin half-maximally inhibited IP3 binding at a concentration of about 3 µM, assuming maximal inhibition of IP3 binding at 10 µM (14, 15). This value is similar to the one found for the purified cerebellar IP3R (14) but is three times higher than that observed for IP3R-1 expressed in Sf9 cells (15). The inhibitory effect of calmodulin was completely abolished at higher pH (Fig. 6B). This was also reported by Patel et al. (14) and is most likely due to an altered conformation of calmodulin at higher pH. High pH is also known to block Ca2+-independent interaction of calmodulin with the ryanodine receptor (35). Our results on calmodulin therefore confirm and extend the results obtained for the intact IP3R-1 by Taylor and co-workers (14, 15) and suggest that a Ca2+-independent interaction site for calmodulin is located in the N-terminal ligand-binding domain of the IP3R-1.


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Fig. 6.   The effect of different [calmodulin] on IP3 binding to Lbs-1. [3H]IP3 binding to partially purified Lbs-1 (2.5 µg) in the presence of the indicated concentrations of calmodulin was expressed as a percentage of the binding measured in Ca2+-free buffer (A, 1 mM EGTA, pH 7.0; B, 1 mM BAPTA, pH 7.8). *, significantly different from binding in Ca2+-free buffer. Data are expressed as the means ± S.E. of at least four experiments, consisting of independent quadriplicates.

Conclusions-- We have expressed the N-terminal 581 amino acids of IP3R-1 containing the complete IP3-binding site in E. coli. Ca2+ dose-dependently inhibited IP3 binding to this protein by decreasing its affinity for IP3. Conceivably, this inhibition is mediated by one of the Ca2+-binding sites that we have previously located within the core IP3-binding pocket of the receptor. Furthermore, calmodulin inhibited IP3 binding to the recombinant ligand-binding site independently of Ca2+. In conclusion, we found functional evidence for both a Ca2+-binding site and a calmodulin-binding site in the N-terminal ligand-binding domain of IP3R-1.

    ACKNOWLEDGEMENTS

We appreciate the skillful technical assistance from Lea Bauwens, Luce Heremans, Yves Parijs, Anja Floorizone, and Marina Crabbé. The p400C1 plasmid containing cDNA of IP3R-1 was kindly provided by Drs K. Mikoshiba and A. Miyawaki (University of Tokyo).

    FOOTNOTES

* This work was supported by grant 3.0238.95 of the Fonds voor Wetenschappelijk Onderzoek, Belgium, and by the Interuniversity Poles of Attraction Program-Belgium State, Prime Minister's Office-Federal Service for Scientific, Technical and Cultural Affairs Grant IUAP P4/23.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Visiting postdoctoral fellow.

§ Senior Research Assistant.

Research Associate of the Foundation for Scientific Research-Flanders, Fonds voor Wetenschappelijk Onderzoek.

parallel To whom correspondence should be addressed. Tel.: 32-16-345725; Fax: 32-16-345991; E-mail: Humbert.DeSmedt{at}med.kuleuven.ac.be.

    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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