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*

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 IP3binding 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 K d 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 μmcalmodulin, 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 μmCa2+ (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.

binding characteristics, and regulation have been identified (1).
Submicromolar [Ca 2ϩ ] inhibits IP 3 binding to cerebellar microsomes (2)(3)(4)(5)(6)(7) and to microsomes of Sf9 insect cells expressing IP 3 R-1 (8,9). Inhibition of IP 3 binding to IP 3 R-1 by Ca 2ϩ might constitute one of the components inducing the descending phase of the bell-shaped dependence of IP 3 -induced Ca 2ϩ release on cytoplasmic Ca 2ϩ (10 -13). There is still controversy about the molecular mechanism responsible for the inhibitory effect of Ca 2ϩ on IP 3 binding, because experiments on purified IP 3 R-1 have given conflicting results. It was suggested that Ca 2ϩ acts directly on a Ca 2ϩ -binding site on IP 3 R-1 (7). On the other hand, indirect inhibition via an accessory Ca 2ϩ -binding protein like calmedin was also reported (4). Moreover, it is still a matter of debate whether Ca 2ϩ -induced inhibition is caused by a decrease in the affinity of IP 3 R-1 for IP 3 (5,7,8) or by a Ca 2ϩ -induced reduction in IP 3 -binding sites (9).
Recently, calmodulin was reported to cause inhibition of IP 3 binding (14,15). Calmodulin would in this case bind to a site different from the Ca 2ϩ -dependent calmodulin-binding site found earlier (16), because calmodulin was able to bind to IP 3 R-1 and inhibit IP 3 binding to IP 3 R-1 in a Ca 2ϩ -independent manner (14,15).
The N-terminal part of the IP 3 R contains all the structural determinants responsible for specific and selective binding of its physiological agonist, IP 3 (17)(18)(19)(20). We have therefore expressed the N-terminal ligand-binding site (first 581 amino acids) of the mouse IP 3 R-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 Ca 2ϩ -binding region located between amino acids 304 -450 (21). We now demonstrate that Ca 2ϩ and calmodulin can both inhibit IP 3 binding to this recombinant protein and that these inhibitors act independently and additively. Our data indicate that the N-terminal ligand-binding domain of IP 3 R-1 contains regulatory regions directly interacting with Ca 2ϩ and calmodulin.

EXPERIMENTAL PROCEDURES
Expression of IP 3 R-1 in Sf9 Insect Cells-The full-length neuronal mouse IP 3 R-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 5Ј-untranslated region of the original p400C1 clone was removed by polymerase chain reaction (PCR) by amplification of the 5Ј-terminal part up to the Cel-II restriction site (nucleotide 555), before subcloning the IP 3 R-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 IP 3 R-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 IP 3 R-1-A bacterial expression vector (pET 21b/ϩ, Novagen Inc.) was used to express the N-terminal 581 amino acids of the mouse IP 3 R-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 IP 3 R-1 (22) as a template. Fragment 1 was produced with forward primer 5Ј-CGCGCATATGTCTGACAAAATGTCG-3Ј (containing an NdeI site including the start codon) and reverse primer 5Ј-CGGAGTATCGATTCATAGG-3Ј (containing the codons for amino acids 64 -65 including a ClaI site). Fragment 2 was synthesized with forward primer 5Ј-GGTCTGTCACGAAGCTTTTGG-3Ј (containing a HindIII site) and reverse primer 5Ј-GTATGCGGCCGCTTACATGAAG-CCAAACTGCTTGG-3Ј (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 pcDNA3.1ϩ (Invitrogen), yielding pcDNA-F2. The SalI digest of p400C1 containing a 329-base pair 5Ј noncoding region and the complete coding region of IP 3 R-1 was first subcloned in the pCI expression plasmid (Promega) to yield pCI-IP 3 R-1. A 1751-base pair fragment was obtained from pCI-IP 3 R-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/ϩ. To remove the 5Ј 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 IP 3 R-1 construct were confirmed by double-stranded sequencing using the Automated Laser Fluorescent TM sequencing system (Amersham Pharmacia Biotech).
Expression in E. coli-The expression of the recombinant N-terminal 581 amino acids of the IP 3 R-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 g/ml ampicillin and grown overnight at 27°C. 1 ml of this culture was diluted in 50 ml of fresh medium (100 g/ml ampicillin) and grown at 21°C for 10 h to an A 600 of about 1.5. Subsequently, expression of the recombinant protein was induced in the presence of isopropyl-1-thio-␤-D-galactopyranoside (0.75 mM) for 20 h at 14°C. Cells were harvested by centrifugation and washed with a buffer containing 10 mM KH 2 PO 4 , 30 mM NaHPO 4 , 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 ␤-mercaptoethanol, 0.8 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, 1 M 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 ϫ g, 60 min), the supernatant containing the soluble fraction of E. coli was stored at Ϫ80°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 l 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.
IP 3 Binding-[ 3 H]IP 3 binding was performed in 100 l of a solution containing 50 mM Tris-HCl, pH 7.0 or 7.8, 50 mM NaCl, 1 mM EGTA or BAPTA, 10 mM ␤-mercaptoethanol, 2.5 g of partially purified Lbs-1, and variable concentrations of [ 3 H]IP 3 (see figure legends) at 0°C for 30 min. Subsequently, 10 l of ␥-globulin (20 mg/ml) and 110 l of 10% polyethylene glycol in IP 3 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 M unlabeled IP 3 . Routinely, specific [ 3 H]IP 3 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 Ͻ 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 IP 3 R-1 was detected with the polyclonal antibody Rbt03. This antibody is directed against the same epitope as the previously described Rbt04 (23)(24)(25)(26) and has specificity and affinity identical to those of Rbt04. A second antibody was raised against the Ca 2ϩ -binding domain cyt3b present between amino acids 378 and 450 of the mouse IP 3 R-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 IP 3 R-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 Sypro TM Orange or transferred to Immobilon-P (Millipore). Blots were blocked for 1 h in a buffer containing 10 mM NaH 2 PO 4 , 32 mM Na 2 HPO 4 , 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 Vistra TM 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). IP 3 was obtained from Roche Molecular Biochemicals. [ 3 H]IP 3 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, ␥-globulins, and heparinagarose beads were obtained from Sigma . Sypro TM Orange was from Bio-Rad. Ethylene glycolbis-(sulfosuccinimidylsuccinate) was from Pierce.

Expression of the Full-length Mouse IP 3 R-1 in Sf9 Insect
Cells-Microsomes of Sf9 insect cells transfected with pVL 1393 IP 3 R-1 (Sf9-IP 3 R-1) were immunoreactive to an antibody specifically recognizing a C-terminal epitope of mouse IP 3 R-1 (Fig. 1A). Under identical conditions, no IP 3 R was detected in control pVL 1393 transfected Sf9 cells. The expressed IP 3 R-1 migrated on SDS-PAGE with the same apparent molecular mass as observed for IP 3 R-1 from rabbit cerebellar microsomes and amounted to 2.5 times the value of the latter (Fig. 1A). In the presence of 5 nM [ 3 H]IP 3 , microsomes of Sf9 cells expressing IP 3 R-1 specifically bound IP 3 . The binding activity (589 Ϯ 83 fmol/mg at pH 7.8) was also about 2.5 times the value found for rabbit cerebellar microsomes (252 Ϯ 43 fmol/mg). Microsomes of control pVL 1393 transfected Sf9 cells showed no significant IP 3 binding under these conditions.
Expression of the IP 3 -binding Domain of the IP 3 R-1-We constructed a bacterial expression vector containing the coding sequence of the N-terminal 581 amino acids of the mouse IP 3 R-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 heparinagarose 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 IP 3 R-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 Sypro TM Orange (data not shown) that at least 80% of the recombinant protein was in the intact form. Degraded proteins missing the primary antibodyepitope (amino acids 378 -450 (21)) or smaller than 40 kDa will not bind IP 3 (20) and will therefore not influence IP 3 binding measurements. The isolated IP 3 -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 crosslinking 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 M IP 3 , 1 M adenophostin-A, or 5 M Ca 2ϩ (data not shown). These results strongly suggest that Lbs-1 is a monomeric polypeptide. 3 Binding to Lbs-1-Fractions of partially purified Lbs-1 specifically bound [ 3 H]IP 3 at a pH of 7.8, whereas a similar fraction from E. coli that was only transformed with host pET 21b/ϩ did not bind IP 3 under these conditions. [ 3 H]IP 3 was displaced from Lbs-1 by unlabeled IP 3 with an IC 50 of 60 nM (Fig. 2). The displacement data could best be fitted using a single-site Scatchard model, yielding a K d of 46 Ϯ 4 nM, a B max of 280 Ϯ 60 pmol/mg, and a Hill coefficient of 1.1 Ϯ 0.1 (Fig. 2, inset). These values are very close to values found for the intact purified mouse cerebellum IP 3 R-1, obtained under similar experimental conditions (IC 50 , 76 nM; Hill coefficient, 1.1 (31); K d , 37 nM (20)). Furthermore, we have previously reported a K d of 46 Ϯ 17 nM for the purified Xenopus IP 3 R-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 IP 3 R-1 showed similar specificity for inositol phosphates and similar affinities for IP 3 as the intact IP 3 R-1. These observations indicate that Lbs-1 is in the right conformation to act as a bona fide IP 3 -binding pocket.

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

FIG. 1. Expression of mouse IP 3 R-1 in Sf9 insect cells and expression of the recombinant IP 3 -binding domain of IP 3 R-1 in E. coli. A, microsomes of Sf9 insect cells transfected with pVL 1393-
Remarkably, the effect of Ca 2ϩ on IP 3 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 Ca 2ϩ -free chelators, espe- A) and partially purified Lbs-1 (2.5 g, B) was measured in the presence or absence of Ca 2ϩ (5 M) and/or calmodulin (10 M) 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 [ 3 H]IP 3 . Sf9-IP 3 R-1 microsomes and partially purified Lbs-1 were pre-exposed to Ca 2ϩ and/or calmodulin for 5 min before adding cially BAPTA, can inhibit IP 3 binding (32,33). The inhibitory effect of Ca 2ϩ on IP 3 binding might therefore be shielded by a stimulatory effect of Ca 2ϩ in relieving inhibition by the chelator. IP 3 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 M free Ca 2ϩ , and 1 mM EGTA (data not shown). Therefore, we can exclude the possibility that the absence of an effect of Ca 2ϩ on IP 3 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 K d value for the binding of IP 3 to pET-581 from 46 to 115 nM. The enhancement of IP 3 binding to the IP 3 R-1 at a higher pH is a well documented phenomenon (5,34). It can be suggested that the different conformational states of the IP 3 R-1 that apparently accompany changes in pH are unequally susceptible to inhibition by Ca 2ϩ .

FIG. 3. Binding of IP 3 to Lbs-1 and microsomes of Sf9-IP 3 R-1 insect cells. [ 3 H]IP 3 binding to microsomes of Sf9-IP 3 R-1 cells (100 g,
Because Lbs-1 was expressed in a bacterial environment, the inhibitory effect of Ca 2ϩ on IP 3 binding to Lbs-1 strongly suggests direct binding of Ca 2ϩ to the N-terminal IP 3 -binding domain of IP 3 R-1 and strongly disfavors the idea of involvement of accessory proteins, such as calmedin (4). The hypothesis of direct binding of Ca 2ϩ to IP 3 R-1 is in agreement with results obtained by Picard et al. (7), who showed that Ca 2ϩ could still inhibit IP 3 binding to rat cerebellar microsomes after removal of peripheral membrane proteins with high alkaline treatment and to immunopurified sheep cerebellar IP 3 R.
We have previously demonstrated direct Ca 2ϩ binding (21) to a stretch of amino acids (304 -450) located in the "core" IP 3binding domain (20). Most likely, Ca 2ϩ exerts its inhibitory effect on IP 3 binding by interacting with this particular region. Our data indicate that the modulation of the IP 3 affinity by cytosolic Ca 2ϩ is an inherent property of the IP 3 -binding domain. Unfortunately, it may be very difficult, if not impossible, to determine the amino acid residues critically involved in the inhibitory effect of Ca 2ϩ on IP 3 binding because mutations and deletions in this region will almost certainly also affect the characteristics of IP 3 binding or eliminate IP 3 binding (20).
Our data give further strong support to the idea that Ca 2ϩ , in a physiological range, is able to inhibit IP 3 binding to IP 3 R-1. This mechanism is likely to contribute to feedback inhibition of IP 3 -induced Ca 2ϩ release by (sub)micromolar [Ca 2ϩ ] (10 -13).
Effect of Calmodulin on IP 3 Binding to Lbs-1-In the presence of Ca 2ϩ , calmodulin binds to the regulatory domain of the type-1 and -2 IP 3 R (16). Recently, it was shown that calmodulin can also bind to IP 3 R-1 in the absence of Ca 2ϩ , thereby inhibiting IP 3 binding to the purified cerebellar IP 3 R (14) and to microsomes of insect Sf9 cells expressing rat IP 3 R-1 (15). In our hands, calmodulin (10 M) inhibited IP 3 binding to microsomes of Sf9 cells expressing IP 3 R-1 and to Lbs-1 by 35.8 Ϯ 8.7 and 43.1 Ϯ 5.9%, respectively (Fig. 3, A and B). These values were similar to maximal inhibition (10 M calmodulin) reported for Sf9 microsomes (40% (15)) and rat cerebellum microsomes (36% (14)). In the presence of both Ca 2ϩ (5 M) and calmodulin (10 M), IP 3 binding was inhibited by 74.5 Ϯ 5.1% (i.e. 50% of the level in the presence of only Ca 2ϩ ). Therefore, inhibition by calmodulin was Ca 2ϩ -independent and was additive to inhibition by Ca 2ϩ , as suggested previously for the intact IP 3 R-1 (14,15). As was also observed for the effect of Ca 2ϩ , inhibition of IP 3 binding by calmodulin does not seem to depend on the presence or absence of the S1 splice domain. The effect of calmodulin on IP 3 binding to Lbs-1 was concentration-dependent (Fig. 6A). Calmodulin half-maximally inhibited IP 3 binding at a concentration of about 3 M, assuming maximal inhibition of IP 3 binding at 10 M (14,15). This value is similar to the one found for the purified cerebellar IP 3 R (14) but is three times higher than that observed for IP 3 R-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 Ca 2ϩ -independent interaction of calmodulin with the ryanodine receptor (35). Our results on calmodulin therefore confirm and extend the results obtained for the intact IP 3 R-1 by Taylor and co-workers (14,15) and suggest that a Ca 2ϩ -independent interaction site for calmodulin is located in the N-terminal ligand-binding domain of the IP 3 R-1.
Conclusions-We have expressed the N-terminal 581 amino acids of IP 3 R-1 containing the complete IP 3 -binding site in E. coli. Ca 2ϩ dose-dependently inhibited IP 3 binding to this protein by decreasing its affinity for IP 3 . Conceivably, this inhibition is mediated by one of the Ca 2ϩ -binding sites that we have previously located within the core IP 3 -binding pocket of the receptor. Furthermore, calmodulin inhibited IP 3 binding to the recombinant ligand-binding site independently of Ca 2ϩ . In conclusion, we found functional evidence for both a Ca 2ϩ -binding site and a calmodulin-binding site in the N-terminal ligandbinding domain of IP 3 R-1.