Molecular Basis of the Isoform-specific Ligand-binding Affinity of Inositol 1,4,5-Trisphosphate Receptors*

Three isoforms of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), IP3R1, IP3R2, and IP3R3, have different IP3-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP3R types exhibited Kd values of 49.5 ± 10.5, 14.0 ± 3.5, and 163.0 ± 44.4 nm, which are close to the intrinsic IP3-binding affinity previously estimated from the analysis of full-length IP3Rs. In contrast, residues 224–604 of IP3R1 and IP3R2 and residues 225–604 of IP3R3, which contain the IP3-binding core domain but not the suppressor domain, displayed an almost identical IP3-binding affinity with a Kd value of ∼2 nm. Addition of 100-fold excess of the suppressor domain did not alter the IP3-binding affinity of the IP3-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP3-binding core domain from different isoforms exhibited IP3-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP3-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP3-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP3-binding affinity of IP3Rs is specifically tuned through the intramolecular attenuation of IP3-binding affinity of the IP3-binding core domain by the amino-terminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP3R isoforms originates from at least the structural difference identified on the suppressor domain.

The inositol 1,4,5-trisphosphate (IP 3 ) 3 receptors (IP 3 Rs) function as IP 3 -gated Ca 2ϩ release channels located on intra-cellular Ca 2ϩ stores, such as the endoplasmic reticulum (1). Mammalian IP 3 R family consists of three isoforms (IP 3 R1, IP 3 R2, and IP 3 R3), and they form homotetrameric or heterotetrameric channels (2). There is evidence of a functional difference among the three isoforms of IP 3 R in terms of their IP 3 sensitivity (3)(4)(5) and cooperativity with respect to IP 3 binding (5). The intrinsic association constants of mouse IP 3 R1, IP 3 R2, and IP 3 R3 expressed in Sf9 cells are estimated to be 3.5 ϫ 10 7 , 1.7 ϫ 10 8 , and 3.4 ϫ 10 6 (M Ϫ1 ), respectively (5). IP 3 R2 exhibits both negative and positive cooperativity, whereas IP 3 R3 exhibits negative IP 3 binding cooperativity (5). This diversity of responsiveness to IP 3 observed among the three IP 3 R isoforms may contribute to the generation of the different degree of IP 3 sensitivity of the Ca 2ϩ store. The molecular basis of the isoform-specific IP 3 -binding affinity, however, is not well understood.
The IP 3 -binding domain of IP 3 R1 is composed of two functional domains, the amino-terminal suppressor domain and the carboxyl-terminal IP 3 -binding core domain (6). The IP 3 -binding core domain is the minimum region required for specific IP 3 binding and is mapped within residues 226 -578 of mouse IP 3 R1, a polypeptide of 2749 residues (6). The amino-terminal 225 amino acid residues of IP 3 R1 function as the suppressor for IP 3 binding, and deletion of these residues results in significant enhancement of IP 3 binding (6). The atomic resolution structures of both the suppressor domain (7) and the IP 3 -binding core domain (8) of mouse IP 3 R1 were solved by x-ray crystallography to 1.8-and 2.2-Å resolution, respectively. The IP 3binding core domain comprises two asymmetric domains, the ␤-domain and ␣-domain. A highly positive-charged pocket is created at the interface of these two domains to which an IP 3 molecule binds. The 11 amino acid residues in the IP 3 -binding core domain of IP 3 R1 are responsible for the coordination of IP 3 (8), and all of them except Gly-268 are conserved in other isoforms. The suppressor domain contains a ␤-trefoil fold and a helix-turn-helix structure inserted between two ␤-strands of the ␤-trefoil fold (7). The conserved 7 amino acid residues, which are clustered on one side of the suppressor domain, were found to be critical for the suppression of IP 3 binding (7). These structural and functional analyses of the IP 3 -binding domain have been carried out mainly using the type-1 isoform, and the suppression ability of the amino-terminal regions of IP 3 R2 and IP 3 R3 has not been well characterized.

Gene Construction-Plasmids
Site-directed mutagenesis within the suppressor domain of T604 m3 or (1-223) m1 -(225-604) m3 (supplemental Fig. S2) to engineer systematic chimeric proteins as summarized in supplemental Fig. S2 was performed with a Quick Change sitedirected mutagenesis kit (Stratagene) and primers containing the appropriate substitution (M1-M13, supplemental Table  S3). Multiple mutants were generated by sequential mutagenesis. Only sense primers used for the site-directed mutagenesis are shown in supplemental Table S3. Substitution of amino acid residues 61-122 of T604 m3 with amino acid residues 62-121 of T604 m1 was carried out using the technique of splicing by overlap extension with PCR (supplemental Table S2) and primers P3, P19, P20, and P21 (supplemental Table S1). The product of the second PCR was digested with NdeI, and the NdeI fragment (0.4 kbp) was replaced with the NdeI fragment (0.4 kbp) of pRSET-T604 m3 . The nucleotide sequences of all of PCR prod- Error bars correspond to the standard deviation. C, comparison between the apparent dissociation constants of T604 and the intrinsic dissociation constants estimated from the measurements using full-length IP 3 Rs expressed in Sf9 cells (5). Circle, IP 3 R1; triangle, IP 3 R2; and square, IP 3 R3. Error bars correspond to the standard deviation.
ucts and site-directed mutants used in this study were confirmed by DNA sequencing with a 3130 Genetic analyzer (Applied Biosystems).
Expression and Purification of Recombinant Proteins-Recombinant proteins were expressed in Escherichia coli BL21codonplus (Stratagene) as described previously (9). Protein purification was performed with a HiTrap heparin HP column (GE Healthcare) according to the method described previously (5). The His 6 -tagged suppressor domain of mouse IP 3 R1 (amino acid residues 2-223) was purified with a ProBond resin (Invitrogen) as described previously (5). Protein concentrations were determined with a protein assay kit (Bio-Rad) and bovine serum albumin as a standard.
IP 3 Binding Assay-An equilibrium IP 3 binding analysis of purified soluble proteins was performed as described previously (9), except for the reaction condition with a cytosol-like medium (110 mM KCl, 10 mM NaCl, 5 mM KH 2 PO 4 , and 50 mM Hepes-KOH, pH 7.4, at 4°C). Purified protein (0.02-0.8 g) was incubated with 0.14 -8.68 nM [ 3 H]IP 3 (New England Nuclear/DuPont) and various concentrations of unlabeled IP 3 (Dojindo) in a binding buffer (cytosol-like medium containing 1 mM dithiothreitol and 0.5 mM EGTA). To avoid tracer depletion, the amount of the protein and the concentration of [ 3 H]IP 3 were adjusted for each experiment. Nonspecific binding was measured in the binding buffer without adding the protein. Nonlinear regression of IP 3 binding data with the Hill-Langmuir equation, where F is the fraction of the recombinant protein that binds IP 3 , [IP 3 ] is the concentration of IP 3 , and K d is the apparent dissociation constant, was performed with Igor Pro (version 4.04, Wavematrics) software.
Modeling of the Suppressor Domain in IP 3 R3-The structure of the IP 3 R1 suppressor domain (residues 7-223) (7) was used for homology modeling of the suppressor domain structure of IP 3 R3, residues 6 -224. The sequence alignment between IP 3 R1 and IP 3 R3 was generated with ClustalW (11) and used for modeling with Modeller (12). Because the loop linking the two helices in the Arm sub-domain of IP 3 R1 (residues 76 -81) was not defined in the original structure, this portion of the suppressor domain was also modeled in the type-3 isoform. Thirty models were calculated and superimposed with Molmol (13) (root mean square deviation ϭ 1.2 Å). Only the mean model is shown.  Fig. 1B is plotted as open circles (B, T604 m1 ; C, T604 m2 ; and D, T604 m3 ). E, comparison of the apparent dissociation constants of (224 -604) m1 , (224 -604) m2 , and (225-604) m3 . Averages of three independent measurements are shown. Error bars correspond to the standard deviation. Statistical analysis was performed using one-way analysis of variance followed by a post-hoc comparison using Dunn's multiple comparison procedure. N.S., not significant.

RESULTS
Characterization of IP 3 Binding to the Amino-terminal 604 Residues of Three IP 3 R Isoforms-Nontagged amino-terminal 604 amino acid residues of the three isoforms of mouse IP 3 Rs, T604 m1 , T604 m2 , and T604 m3 (supplemental Fig. S1), were expressed in E. coli and were purified on a HiTrap heparin HP column. Recombinant T604 proteins showed an apparent molecular mass of ϳ65 kDa (Fig. 1A). We measured the IP 3binding activity of these purified proteins using 3 H-labeled IP 3 . Fig. 1B shows the relationship between the normalized amount of IP 3 bound to the purified proteins and the IP 3 concentration applied. Because the data of the three isoforms were well fitted with the Hill-Langmuir equation (see "Experimental Procedures") and each protein possesses a single IP 3 -binding site, IP 3 binding to the purified amino-terminal 604 amino acid residues seemed to occur independently. The apparent dissociation constants of T604 m1 , T604 m2 , and T604 m3 were estimated to be 49.5 Ϯ 10.5 nM (n ϭ 3), 14.0 Ϯ 3.5 nM (n ϭ 3), and 163.0 Ϯ 44.4 nM (n ϭ 9) (mean Ϯ S.D.), respectively. These values are well consistent with the intrinsic dissociation constants estimated from the analyses of homotetrameric IP 3 R channels expressed in Sf9 cells under the same experimental condition (28.6 nM for IP 3 R1, 5.9 nM for IP 3 R2, and 294.0 nM for IP 3 R3) (5) (Fig. 1C). These results suggest that the type-specific IP 3 -binding affinity originates from the amino-terminal 604 amino acid residues of each IP 3 R type.
Comparison of the IP 3 -binding Affinity of the IP 3 -binding Domain of Three Isoforms without the Suppressor Domain-The IP 3 -binding domain of IP 3 R1 is functionally divided into two parts: the amino-terminal suppressor domain and the carboxylterminal IP 3 -binding core domain (6,14). The IP 3 -binding core domain has been defined as a minimum essential region for specific IP 3 binding, which resides within amino acid residues 226 -578 of IP 3 R1 (6). Because the amino acid residues 224 -604 of mouse IP 3 R1, designated as (224 -604) m1 (supplemental Fig. S1B), are expressed well in E. coli (9), we used it for the measurement of the IP 3 -binding affinity of the IP 3 -binding domain without the suppressor domain. Supplemental Fig. S1A shows portions of the amino acid sequence alignments among the three mouse IP 3 R isoforms. To compare the unsuppressed IP 3 -binding affinity among the three isoforms, we purified the bacterially expressed (224 -604) m1 , residues 224 -604 of mouse IP 3 R2 ((224 -604) m2 ), and the residues 225-604 of mouse IP 3 R3 ((225-604) m3 ) ( Fig. 2A). We found that all of the IP 3 binding data of these proteins were well fitted with the Hill-Langmuir equation (Equation 1) (Fig. 2, B-D) with statistically indistinguishable apparent dissociation constants, 1.78 Ϯ 0.63 nM (n ϭ 3) for (224 -604) m1 , 1.51 Ϯ 0.01 nM (n ϭ 3) for (224 -604) m2 , and 2.04 Ϯ 0.65 nM (n ϭ 3) for (225-604) m3 (mean Ϯ S.D.) (Fig. 2E). These results indicate that the presence of the amino-terminal 223, 223, and 224 amino acid residues of mouse IP 3 R1, IP 3 R2, and IP 3 R3 results in the suppression of IP 3 binding by 27.8-fold (Fig. 2B), 9.3-fold (Fig. 2C), and 81.1-fold (Fig. 2D), respectively. Moreover, the isoform-specific IP 3 -binding affinity of the native IP 3 Rs reflects the different degree of the suppression of IP 3 binding by the suppressor domain of each isoform.
Mechanism of IP 3 Binding Suppression-To assess the mechanism for the suppression, we investigated the effect of the addition of the bacterially expressed His 6 -tagged amino acid residues 2-223 of mouse IP 3 R1 (H(2-223) m1 ) (supplemental Fig. S1) on the IP 3 -binding activity of (224 -604) m1 . As shown in supplemental Fig. S3 the addition of 100-fold excess (molar ratio) purified H(2-223) m1 did not significantly alter the IP 3binding affinity of (224 -604) m1 . These results indicate that the suppression of IP 3 binding requires a covalent bond between the suppressor domain and the IP 3 -binding core domain.
Functional Difference among the Suppressor Domains of the Three IP 3 R Isoforms-We created six chimeric proteins composed of a suppressor domain fused with an IP 3 -binding core domain from different isoforms (supplemental Fig. S1B) to analyze the mechanism underlying the generation of isoform-specific IP 3 -binding affinity. All of the equilibrium IP 3 binding data obtained for these chimeric proteins showed good fits with the  Fig.  3. All of the chimeric proteins showed Ͼ10-fold lower IP 3binding affinity (Fig. 3, A-C) compared with the proteins that do not possess the suppressor domain (K d Ϸ 2 nM) (Fig. 2). The artificial chimeric proteins, however, revealed apparent dissociation constants that significantly differed from those of the proteins composed of the native combination of the suppressor domain and the IP 3 -binding core domain (Fig. 3, A-C). This suggests that the suppressor domain alone is not a prime determinant of the affinity for IP 3 . When we compare the same data in respect of the IP 3 -binding core domain (Fig. 3, D-F), the different character of the IP 3 -binding core domain of the three isoforms became obvious. The IP 3 -binding core domain of the type-1 isoform exhibited an almost identical IP 3 -binding affinity despite the type of the suppressor domain connected (Fig.  3D). The type-2 IP 3 -binding core domain showed indistinguishable affinity for IP 3 when it was fused with the type-1 and type-2 suppressor domains, but the chimeric protein with the type-3 suppressor domain showed an affinity ϳ3-fold lower compared with the native combination of the type-2 isoform (Fig. 3E). The IP 3 -binding core domain of the type-3 isoform strictly required the type-3 suppressor domain to generate its native IP 3 -binding affinity (Fig. 3F). These results indicate that: 1) the isoform-specific IP 3 -binding affinity is predominantly determined by the IP 3 -binding core domain rather than the suppressor domain; 2) the suppressor domains of the type-1 isoform and type-2 isoform are mutually interchangeable for both the IP 3 -binding core domains of IP 3 R1 and IP 3 R2 to generate their native IP 3 -binding affinity; and 3) the type-3-specific site(s) in the suppressor domain may be critical for the proper suppression of the IP 3 -binding core domain of IP 3 R3. 3 R3-To elucidate the structural basis for the suppression of the type-3 isoform, we created a series of mutated proteins based on the amino acid sequence difference between IP 3 R1 and IP 3 R3 and on the three-dimensional structure of the suppressor domain of IP 3 R1 (supplemental Fig. S2). The suppressor domain forms a hammer-like structure with the head subdomain and the arm subdomain (7). The head subdomain consists of two single-turn ␣-helices and 12 ␤-strands, which form a ␤-trefoil fold (7). The arm subdomain is made of a helixturn-helix structure, which is inserted between the fourth and fifth ␤-strands of the head subdomain (7). We hypothesized that the critical regions for the suppression of the type-3 isoform are located within the 11 loop segments that connect ␤-strands (Figs. 4A and S2), because in the case of ␤-trefoil fold architecture most of the surface properties are dictated by the residues from the loop segment, rather than the residues comprising the barrel and the triangular array of the core structure (15). To evaluate this hypothesis, we first constructed a mutant protein in which all of the non-conserved amino acid residues within the ␤-strand regions of T604 m3 were substituted with residues appearing in IP 3 R1 (supplemental Fig. S2). Non-conserved residues are located within the first, fourth, eighth, ninth, and twelfth ␤-strand regions (␤1, ␤4, ␤8, ␤9, and ␤12, respectively, supplemental Fig. S2) (7). The mutant protein, (␤1,␤4,␤8,␤9,␤12) m1 /T604 m3 , exhibited an apparent dissociation constant of 254.0 Ϯ 48.5 nM (n ϭ 3), which was indistinguishable from that of T604 m3 (163.0 Ϯ 44.4 nM) (n ϭ 9) (Fig.  4B). These results indicate that the critical sites for type-3-specific suppression are located within the loop regions, but not within the ␤-strand regions, of the suppressor domain. Type-3specific amino acid sequences are located within the first, third, fourth, fifth, seventh, eighth, and tenth loop regions (L1, L3, L4, L5, L7, L8, and L10, respectively, supplemental Fig. S2) (7).

DISCUSSION
The IP 3 sensitivity of the intracellular Ca 2ϩ stores seems to be heterogenous within the cytosol, and spatially restricted Ca 2ϩ releases, such as Ca 2ϩ puffs, have frequently been observed in various cell types (16,17). Multiple intracellular Ca 2ϩ stores with variable IP 3 sensitivities in single cells may contribute to the generation of the complex spatiotemporal patterns of intracellular Ca 2ϩ dynamics (18 -21). We have previously reported that three isoforms of the IP 3 R have different IP 3 -binding affinities and cooperativities (5). A non-cooperative IP 3 binding model did not fit the equilibrium IP 3 binding data obtained from tetrameric IP 3 R2 or IP 3 R3 complexes expressed in Sf9 cells (5), indicating that the IP 3 -binding affinity of vacant subunits within single tetrameric complexes changes depending on the number of occupied subunits within a homotetrameric complex composed of IP 3 R2 or IP 3 R3. The intrinsic IP 3 -binding affinities, that is the IP 3 -binding affinities of four vacant subunits within single non-liganded homotetrameric complexes, have been estimated to be 3.5 ϫ 10 7 M Ϫ1 for IP 3 R1, 1.7 ϫ 10 8 M Ϫ1 for IP 3 R2, and 3.4 ϫ 10 6 M Ϫ1 for IP 3 R3 (5). In the present study, we analyzed the molecular basis of the isoform-specific intrinsic IP 3 -binding affinity of IP 3 R. The amino-terminal 604 amino acid residues of the three isoforms exhibited almost the same IP 3 -binding affinities with the intrinsic IP 3 -binding affinities estimated from full-length IP 3 Rs expressed in Sf9 cells (Fig. 1). These results indicate that the intrinsic IP 3 -binding affinity of homotetrameric IP 3 R channels is predominantly determined by the amino-terminal 604 amino acid residues of each subunit. The amino-terminal 225 residues have been known to reduce the IP 3 -binding affinity of the IP 3 -binding core domain of IP 3 R1 by Ͼ10-fold (6), and the deletion of the amino-terminal 225 residues has been reported to enhance the IP 3 -binding activity of IP 3 R3 (22); however, the effect of the suppressor domain on the affinity of the IP 3 -binding core domain has never been quantitatively characterized for type-2 and type-3 isoforms. We measured the IP 3 -binding affinity of residues 224 -604 of IP 3 R2 and residues 225-604 of IP 3 R3 and found that both proteins showed an IP 3 -binding affinity higher than those obtained for residues 1-604 of IP 3 R2 and IP 3 R3 by 9.3-and 81.1-fold, respectively (Fig. 2). Interestingly, in the absence of the suppressor domain, all three isoforms exhibit a nearly identical IP 3 -binding affinity (Fig. 2). Because Bultynck et al. (23) has shown that the suppressor domain of IP 3 R1 (residues 1-225) can physically interact with the IP 3 -binding core domain of IP 3 R1 (residues 226 -604), the interdomain interaction may cause changes in the IP 3 -binding affinity of the IP 3 -binding core domain. The amino-terminal 604 residues of three IP 3 R types bind IP 3 in a non-cooperative manner (Fig. 1B), indicating that the suppression of IP 3 binding occurs independently in each molecule. Because the addition of a 100-fold excess of the suppressor domain did not significantly alter the IP 3 -binding affinity of the IP 3 -binding core domain (supplemental Fig. S3), the suppressor domain is not effective for separating molecules. We therefore suspect that the suppression of IP 3 binding is an intramolecular event that requires a covalent bond between the suppressor domain and the IP 3 -binding core domain. The present results suggest that 1) the isoform-specific IP 3 -binding affinity originates from the intramolecular suppression of IP 3 binding, but not from the difference in the intrinsic IP 3 -binding affinity of the IP 3 -binding core domain, and 2) the nature of the interaction between the suppressor domain and the IP 3 -binding core domain is different among the three isoforms, thereby resulting in their different IP 3 -binding affinities. In other words, the IP 3 -binding affinity of IP 3 Rs is generated by the precisely controlled attenuation of IP 3 -binding affinity caused by the intramolecular interaction between the suppressor domain and the IP 3 -binding core domain, and the IP 3 -binding affinity is a tunable parameter rather than a stable constant.
Molecular Basis of the Type-specific Suppression of IP 3 Binding-To test domain compatibility among the three IP 3 R types, we have engineered chimeric proteins in which the amino-terminal suppressor domain was fused to the IP 3binding core domain from different isoforms and found that the suppressor domain is not a prime determinant of the IP 3 -binding affinity (Fig. 3, A-C). The IP 3 -binding core domain of IP 3 R1 showed almost the same apparent dissociation constants regardless of the type of the suppressor domain connected (Fig. 3D). The IP 3 -binding core domain of IP 3 R2 did not discriminate the suppressor domain from IP 3 R1 and IP 3 R2, but the suppressor domain from IP 3 R3 failed to produce the native IP 3 -binding affinity of IP 3 R2 (Fig. 3E). The IP 3 -binding core domain of IP 3 R3 strictly required the suppressor domain from the same isoform to produce its native IP 3 -binding affinity (Fig. 3F). These results indicate that 1) conserved amino acid residues in the suppressor domain of IP 3 R1 and IP 3 R2 are involved in the interaction with the type-1 and type-2 IP 3 -binding core do- between the suppressor domain and the IP 3 -binding domain. According to the prediction, the essential amino acid residues for the generation of the type-3 receptor-specific IP 3 -binding affinity are Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179 (Figs. 6 and 7). Szlufcik et al. showed that the deletion of amino acid residues 76 -86 of IP 3 R1 or the replacement of these residues by residues 75-86 of IP 3 R3 resulted in an increase in IP 3 binding and the sensitivity of IP 3 -induced Ca 2ϩ release (22). In our experiments, the replacement of L4, which includes residues 76-86 of IP 3 R1 or residues 75-86 of IP 3 R3, did not induce a significant change in the apparent dissociation constant of T604 m3 (Fig. 5A). Because Szlefcik et al. (22) measured the amount of [ 3 H]IP 3 bound to crude membrane fractions prepared from cell lines with different expression levels of recombinant receptors, it is not clear whether the mutations on residues 76 -86 significantly affected the IP 3 -binding affinity of the actual receptor proteins. Because our results reported here are mainly based on an analysis of the IP 3 -binding domain fragments expressed in bacteria, the critical sites for determining isoform-specific IP 3 -binding affinity in full-length receptor proteins should be confirmed. Identification of the interface between the suppressor domain and the IP 3 -binding core domain by x-ray or NMR analyses should help our better understanding of the nature of the molecular interaction between these two domains.
A Possible Mechanism for the Suppression of IP 3 Binding-Addition of 100-fold excess of the type-1 suppressor domain did not result in a significant reduction of the IP 3 -binding affinity of the type-1 IP 3 -binding core domain (supplemental Fig.  S3). This result indicates that the suppression of IP 3 binding is not a simple competitive inhibition of IP 3 binding to the IP 3binding core domain by the suppressor domain. Recently, we have measured the reaction kinetics of the fluorescence resonance energy transfer of the IP 3 sensor protein upon the IP 3 binding (24). The IP 3 sensor protein, designated IRIS, is composed of the type-1 IP 3 -binding core domain fused to two fluorescent proteins, enhanced cyan fluorescent protein and Venus. Analysis of the reaction kinetics of IRIS shows that there are at least two conformational states of the IP 3 -binding core domain in the absence of IP 3 and that IP 3 binding fixes the conformation of the IP 3 -binding core domain to one state (24). This reaction model is consistent with the result of the NMR studies, which suggests a dynamic equilibrium exists between two or more conformations in the apo state of the IP 3 -binding core domain (25). According to this model, not only the rate constants for ligand-association and dissociation but also the rate constants for conformational changes of the receptor protein determine the equilibrium dissociation constant between the receptor and ligand. Evaluation of the effect of the suppressor domain on the rate of ligand binding and the rate of conformational changes of the IP 3 -binding domain should help us to understand the mechanism of the IP 3 binding suppression.
Functional Significance of the Intramolecular Turning of IP 3binding Affinity-What is the functional significance of the intramolecular attenuation of the intrinsic IP 3 -binding affinity of IP 3 Rs except for the generation of the isoform-specific IP 3binding affinity? We have previously shown that the IP 3 binding to the tetrameric complex of IP 3 R2 or IP 3 R3 is not a random process, and the occupation of the IP 3 -binding site changes the IP 3 -binding affinity of vacant sites on the neighboring subunits (5). During the sequential binding of four IP 3 molecules to single tetrameric complexes of IP 3 R2 and IP 3 R3, the dissociation constants of vacant sites are estimated to be changed to 5.8 ϫ 10 Ϫ9 M (0 IP 3 molecule/tetramer), 3.7 ϫ 10 Ϫ8 M (1 IP 3 molecule/tetramer), 1.3 ϫ 10 Ϫ6 M (2 IP 3 molecules/tetramer), and 3.4 ϫ 10 Ϫ7 M (3 IP 3 molecules/tetramer) for IP 3 R2 and to 2.9 ϫ 10 Ϫ7 M (0 IP 3 molecule/tetramer), 7.0 ϫ 10 Ϫ7 M (1 IP 3 molecule/tetramer), 8.2 ϫ 10 Ϫ7 M (2 IP 3 molecules/tetramer), and 2.8 ϫ 10 Ϫ6 M (3 IP 3 molecules/tetramer) for IP 3 R3, respectively (5). These flexible changes of IP 3 -binding affinity may be generated from the molecular interaction between occupied subunits and vacant subunits within a single tetrameric channel complex through the suppressor domain of vacant subunits. We have also pointed out that the suppressor domain is a focal point for the interaction with various modulatory proteins, including calmodulin, CaBP1, and RACK1 (7). These proteins may have some potential for the modulation of the IP 3 -binding affinity of IP 3 Rs by changing the degree of the suppression of IP 3 binding. Our recent analysis of cytosolic IP 3 dynamics in single living cells suggest that IP 3 sensitivity of the intracellular Ca 2ϩ stores continuously change during Ca 2ϩ oscillations (24). The unique ligand binding machinery, composed of the suppressor domain and the IP 3 -binding core domain of IP 3 Rs, may account for the generation of the dynamic change of the sensitivity of the receptor for IP 3 in living cells.