Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.

To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding “core.” Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Many cellular responses to hormones, neurotransmitters, growth factors, etc. are mediated by the intracellular second messenger inositol 1,4,5-trisphosphate (IP 3 or (1,4,5)IP 3 ) 1 (1). IP 3 releases Ca 2ϩ from intracellular stores by binding to the IP 3 receptor (IP 3 R) (2), which is a tetrameric IP 3 -gated Ca 2ϩ release channel (3)(4)(5). There are at least three types of IP 3 R derived from distinct genes in mammals (6 -12). Structural and functional studies on type I IP 3 R (IP 3 R1) (2749 amino acids, 313 kDa) have revealed that it is structurally divided into three parts: a large N-terminal cytoplasmic arm (83% of the receptor molecule); a putative six membrane-spanning domains clustered near the C terminus, which are thought to constitute an ion channel by forming a tetramer; and a short C-terminal cytoplasmic tail (13,14).
The binding of IP 3 to this receptor purified from mouse cerebella is stoichiometric (K d ϭ ϳ100 nM, Hill coefficient ϭ ϳ1.0) (2,15). To localize the IP 3 binding site, deletion mutagenesis studies showed that IP 3 R1 binds IP 3 within the N-terminal 650 amino acids independently of the tetramer formation (16,17). Newton et al. (18) have reported that the N-terminal 576 amino acids fused to glutathione S-transferase specifically bound IP 3 with high affinity, whereas further N-or C-terminal deletions of this region completely abolished the specific binding. Furthermore, Mourey et al. (19) have reported that residues 471-501 in this region were labeled with a photoaffinity ligand. These results indicated that the IP 3 binding site is localized within the N-terminal 576 amino acids and consists of some distantly separated motifs. IP 3 is characterized by three negatively charged phosphate groups at equatorial positions 1, 4, and 5 of an inositol ring. Ca 2ϩ release experiments using various synthetic inositol phosphate analogues showed that the IP 3 recognition site is markedly stereospecific (20 -22). The ability of IP 3 to release Ca 2ϩ depends critically upon the positional distribution of the phosphate groups around the inositol ring, suggesting that binding sites for these three phosphate groups make major contributions to the recognition and binding of (1,4,5)IP 3 . Thus, it has been assumed that there is a pocket of positive charges that facilitate ionic interactions with the negative charges on these three phosphate groups. This hypothetical model is supported by the following evidence. IP 3 binding to the platelet membrane is blocked by the specific Arg-modifying reagent, p-hydroxyphenylglyoxal, suggesting the involvement of Arg in the IP 3 binding (23). IP 3 binding to the receptor is competitively blocked by heparin (24), and the IP 3 R protein has been purified by heparin affinity column chromatography (2,15).
The known binding site for heparin in antithrombin III is highly basic because of enriched Arg or Lys residues (25). The IP 3 binding to the receptor is augmented with increasing pH over the range 5-9 (24). A study using NMR spectroscopy showed that IP 3 dissociates protons from three phosphate groups over this pH range, indicating that the negative charges of IP 3 contribute its binding to the receptor (26). Finally, x-ray crystallographic studies of the pleckstrin homology domain of ␤ spectrin and phospholipase C-␦, which bind IP 3 but have no sequence homology with IP 3 R, in complex with IP 3 showed that 2 and 5 positively charged amino acid residues of this domain, respectively, interact with the phosphate groups of IP 3 (27,28).
Despite the above evidence, the detailed molecular structure of the IP 3 binding site of the IP 3 R remains to be studied. In this study, we developed an Escherichia coli expression system for various recombinant IP 3 binding sites of mouse IP 3 R1. The N-terminal 734 amino acids of IP 3 R1 (T734) expressed in E. coli exhibited similar binding characteristics to those of the native cerebellar IP 3 R. Eighteen deletion mutageneses of T734 showed that 353 amino acids (residues 226 -578) are directly responsible for the IP 3 binding. Furthermore, we performed site-directed mutagenesis on 41 basic amino acid residues within the N-terminal 650 amino acids of T734 and showed that 10 single amino acid substitutions markedly reduced the IP 3 binding activity. They were scattered within residues 226 -578 and conserved among all members of the IP 3 R family. Of these, three were critical for IP 3 binding and one was involved in binding specificity. We discuss the structure of the IP 3 binding site of the IP 3 R.

EXPERIMENTAL PROCEDURES
Materials-Recombinant Pfu DNA polymerase, Taq DNA polymerase, and restriction and other modification enzymes were obtained from Stratagene, Takara Shuzo (Otsu, Japan), and New England Biolabs.
Plasmid Construction and Mutagenesis-To clone the cDNA encoding the N-terminal 734 amino acids of mouse IP 3 R1 into the pET-3a vector (29) in frame, a site for NdeI (CATATG) was introduced into the first codon (underlined; nucleotide position 329) of the cDNA by sitedirected mutagenesis using an oligonucleotide (5Ј-TGTCAGACATAT-GCGTGTTGGAA-3Ј, nucleotide position 338 -316) and a Mutan-K kit (Takara Shuzo). The pUC 119 vector containing a cDNA fragment from nucleotide positions 147 to 1,029 (5T9 E2-1 (6)) was the template for mutagenesis. The SacI-ClaI (nucleotide position 147-521) fragment containing the mutagenized sequence and the ClaI-EcoRV (nucleotide position 521-2,532) fragment from pBactS-C1 (17) were ligated into the SacI-SmaI sites of pUC119 vector. From the resultant plasmid, the NdeI (introduced into nucleotide position 328)-BamHI (present in pUC119) fragment was subcloned into the NdeI-BamHI sites of pET-3a vector. The plasmid designated pET-T734 encoded the N-terminal 734 amino acids of mouse IP 3 R1 followed by 5 additional amino acids (WGSGC) derived from pUC 119 at the C terminus.
Expression of Recombinant Ligand Binding Site in E. coli-A single colony of E. coli BL21(DE3) (29) transformed with pET-T734 was selected into 1.5 ml of Luria-Bertani medium containing 100 g/ml ampicillin and incubated at 27°C for 13-14 h (full growth). One hundred microliters of the culture was inoculated in 10 ml of Luria-Bertani medium containing 100 g/ml ampicillin and incubated at 21°C for 9-10 h to an A 600 of ϳ1.5. After addition of isopropylthio-␤-D-galactoside to a final concentration of 0.5 mM, the culture was incubated at 14°C for another 20 h. Cells were harvested by centrifugation, washed with 1 ml of phosphate-buffered saline, and stored at Ϫ80°C. The same procedures were performed on E. coli cells harboring other mutant plasmids or pET-3a.
Preparation of Soluble Fraction of E. coli-Cell pellets were resuspended in 1 ml of binding buffer (50 mM Tris⅐HCl (pH 8.0 at 4°C), 1 mM 2-mercaptoethanol, 1 mM EDTA) containing protease inhibitors (10 M pepstatin A, 10 M leupeptin, and 0.6 mM phenylmethylsulfonyl fluoride), incubated with 0.1 mg/ml of lysozyme at 4°C for 30 min, then subjected to six cycles of freezing in liquid nitrogen followed by thawing in a water bath at 37°C. Chromosomal DNAs were sheared with a sonicator (Astrason XL2020) in the presence of 32 g/ml DNase I for 10 s at 4°C twice. The suspension was centrifuged at 30,000 ϫ g for 60 min at 2°C. The supernatant was Western blotted and IP 3 binding was assayed after the protein concentration was determined using a kit (Bio-Rad) with bovine serum albumin as the standard.
Polyclonal Rabbit Antiserum Preparation-A polyclonal antiserum was raised in a rabbit to a synthesized peptide corresponding to residues 501-518 of mouse IP 3 R1, which was coupled via an additional N-terminal cysteine to keyhole limpet hemeocyanin using the crosslinking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester.
[ 3 H]IP 3 Binding Assay-Soluble protein (30 g) was incubated with 9.6 nM [ 3 H]IP 3 (or 0.96 nM for Scatchard analyses of the expressed residues 224 -579) in 100 l of binding buffer for 10 min at 4°C. The mixture was then added to 4 l of ␥-globulin (50 mg/ml) and 100 l of a solution containing 30% (w/v) polyethylene glycol 6000, 50 mM Tris⅐HCl (pH 8.0 at 4°C), 1 mM 2-mercaptoethanol, and 1 mM EDTA. After incubation at 4°C for 5 min, the protein-polyethylene glycol complex was collected by centrifugation at 10,000 ϫ g for 5 min at 2°C. We confirmed by Western blotting that all expressed recombinant proteins were precipitated under these conditions (data not shown). The pellets were dissolved in 180 l of Solvable (DuPont NEN). After neutralization with 18 l of acetic acid, the radioactivity was measured in 5 ml of Atomlight (DuPont NEN) with a liquid scintillation counter. The specific binding was defined by subtracting the nonspecific binding in the presence of 2 or 10 M of cold IP 3 from the total. Specific [ 3 H](1,4,5)IP 3 binding was also inhibited in the reaction mixture described above, except for the presence of various inositol phosphates.

RESULTS
Characterization of T734 Expressed in E. coli-To define the structural determinants of the IP 3 binding site of IP 3 R1, we developed an E. coli expression system for the N-terminal 734 amino acids (named T734) that contain the IP 3 binding region (16 -18) and the epitope of anti-IP 3 R1 mAb 4C11 (residues 678 -699) (6). The constructs were designed so that expressed recombinant proteins had no foreign sequences, such as fusion proteins, to avoid interference with the IP 3 binding, except for the 5 additional amino acids at the C terminus. The recombinant proteins expressed in E. coli were almost all found in aggregates (inclusion bodies) when cells were grown at 37°C, as judged by Western blotting using mAb4C11. We did not detect any significant IP 3 binding activity in the soluble fraction. On the other hand, when cells were grown at 14 -28°C, some fractions of the expressed proteins became soluble. A specific immunoreactive band of about 80 kDa was detected by mAb4C11 in the soluble fraction from T734-expressing cells (Fig. 1A). The soluble fraction from T734 cells grown at the low temperature bound significantly high levels of IP 3 compared with the control cells, which showed no activity (Fig. 1B). Thus, the IP 3 binding properties of the soluble fraction containing T734 were further characterized without purification. The binding specificity of T734 to various inositol phosphates was examined by competition for [ 3 H](1,4,5)IP 3 binding (Fig. 1C). The competitive potency was in the following order: (1,4,5)IP 3 Ͼ (2,4,5)IP 3 Ͼ (1,3,4,5)IP 4 Ϸ (4,5)IP 2 . (1,4)IP 2 and (1)IP 1 did not compete, even at a concentration of 10 M. This binding specificity was very similar to that of the native IP 3 R from mouse cerebellum (data not shown). Scatchard analysis indicated that the dissociation constant (K d ) of the IP 3 binding to T734 was 50 Ϯ 2.4 nM (n ϭ 6) (Fig. 1D), which was also consistent with that of the cerebellar IP 3 R (37 nM). When the soluble fraction from T734-expressing cells was applied to a heparin-agarose column, the T734 protein was retained on the column in low salt (0.25 M NaCl) and was eluted in high salt (0.5 M NaCl) (data not shown). The IP 3 binding activity of T734 was completely inhibited by heparin, as was cerebellar IP 3 R. These results demonstrated that the bacterially expressed T734, even in the crude soluble fraction, had similar IP 3 binding characteristics to those of cerebellar IP 3 R.
Determination of IP 3 Binding Site by Deletion Mutagenesis-To define the boundary of the IP 3 binding site, we constructed 2 internal deletion mutants and 15 N-terminal deletion mutants based on the T734 construct ( Fig. 2A). Deletion mutants were named "D(deleted amino acid positions)." We confirmed the expression of these mutant proteins by Western blotting using mAb4C11 (Fig. 2C). As shown in Fig. 2B, one of the internal deletion mutants, D(579 -649) retained IP 3 binding activity, whereas that with a further 11-amino acid deletion, D(568 -649), completely lost the activity, indicating that the C-terminal boundary of the binding site was located be- Characterization of the 356-Amino Acid Polypeptide (Residues 224 -579) Close to the IP 3 Binding Core-We expressed the 356-amino acid polypeptide (residues 224 -579), which appeared to be about the minimum required for specific IP 3 binding. An expressed recombinant protein of approximately 42 kDa was detected using polyclonal rabbit antiserum raised to a synthesized peptide corresponding to amino acids 501-518 of mouse IP 3 R1 (Fig. 3A). The soluble fraction containing this 42-kDa protein exhibited significant IP 3 Fig. 3C, this 42-kDa IP 3 binding protein exhibited similar binding specificity to that of T734 ((1,4,5)IP 3 Ͼ (2,4,5)IP 3 Ͼ (1,3,4,5)IP 4 ). The K d value of this protein was 2.3 nM, which was over 10-fold lower than that of T734 (Fig. 3D). These results indicated that residues 224 -579 were sufficient for specific and high affinity binding to IP 3 . Together with the data from D(1-225) and D(579 -649), this indicates that the critical site for specific IP 3 binding is located between the residues 226 -578.

3B). As shown in
Determination of Critical Amino Acids by Site-directed Mutagenesis-Site-directed mutagenesis of the Arg and Lys residues was performed to identify the amino acids involved in the IP 3 binding, since it has been assumed that positively charged amino acid residues are responsible for binding to negatively charged IP 3 (20). Fig. 4 shows the amino acid sequence of the N-terminal 650 amino acids of mouse IP 3 R1, containing the IP 3 binding site (17). Among 84 positively charged residues (37 Arg and 47 Lys residues) scattered within this region, we selected 41 for site-directed mutagenesis according to the following criteria. The critical amino acids for the IP 3 binding are probably localized within residues 226 -578 as shown above, and probably conserved among all members of the IP 3 R family. Thus, we mutated all of the conserved Arg and Lys residues within residues 226 -578 (Arg-241, 265, 269, 293, 304, 441, 470, 471, 504, 506, 511, 537, 545, 554, 558, 561, and 568 and Lys-235, 249, 306, 424, 427, 459, 508, 569, and 576). We also mutated Arg-257 and 376 and Lys-258, 259, 350, 412, and 501, which are not aligned at identical positions among all IP 3 Rs for maximal homology, but they seem to be conserved if spacing is considered (a shift of one position to either side). In addition, we mutated Arg-603, 623, 626, and 629 and Lys-100, 101, 604, and 624, which are conserved (except for Arg-626 and Lys-100) and are located in regions with putative high surface probability, which seemed to be implicated in the IP 3 binding. All these residues were substituted with a neutral amino acid, Gln, either singly or in pairs. The site-directed mutants were named mutated amino acid residues (one-letter amino acid notation ϩ position) plus the substituted amino acid (one-letter amino acid notation), e.g. K100Q/K101Q, a mutant having double substitution of Gln for Lys at positions 100 and 101. The specific IP 3 binding activity of site-directed mutants was normalized with that of the wild-type T734, considering their expression level, which we quantified by Western blotting using mAb4C11 followed by densitometry (Fig. 5). Site-directed mutants were divided into four groups on the basis of their IP 3 binding activity: none (less than 5% activity of the wild type; R265Q, R504Q/R506Q, K508Q, R511Q, and R568Q/K569Q), partial (5-30% activity of wild type; R241Q, K249Q, R269Q, R504Q, R506Q, R568Q, and K569Q), enhanced (more than 200% activity of the wild type; R441Q) and unchanged (K100Q/K101Q, Although the single amino acid substitution mutants (R504Q, R506Q, R568Q, and K569Q) retained partial binding activity, double mutants (R504Q/R506Q and R568Q/K569Q) completely lost it (Fig. 5).
Mutagenesis of Lys-508, Arg-511, and Lys-569 to Ala or the Other Basic Amino Acid Residues-To rule out possibility that Gln substituted for Arg or Lys itself affects the IP 3 binding activity, Lys-508, Arg-511, and Lys-569 were substituted with the small neutral residue, Ala. As shown in Table I, both K508A and R511A completely lost IP 3 binding activity, and K569A did partially. These data agree with those obtained from their Gln mutation counterparts. Therefore, the mutational effects shown in Fig. 5 are not attributable to the substituted Gln itself.
To further verify these findings, we mutated Lys-508, Arg-511, and Lys-569 to other positively charged residues (Lys3 Arg and Arg3 Lys) ( Table I). The mutants K508R and R511K completely lost the activity. The other mutant, K569R, retrieved a little but retained only ϳ20% activity of T734. As a result, even these functionally conservative mutations markedly reduced the IP 3 binding activity.

DISCUSSION
It has been assumed that IP 3 R has a pocket with a highly restricted structure that specifically recognizes the IP 3 molecule (20). What is the minimum number of amino acids required to assure the binding conformation, and which residues are present on the surface of the pocket? To address these questions by molecular biological means, we developed an E. coli expression system in which the N-terminal 734-amino acid residues of mouse IP 3 R1 (T734) are expressed as soluble proteins. The binding affinity for (1,4,5)IP 3 and specificity for various inositol phosphates of the expressed T734 proteins were similar to those of cerebellar IP 3 R, indicating that the binding sites expressed in this system form a functional conformation resembling the native binding site.
Structure of the IP 3 Binding Site (Residues 226 -578)-We found that, at most, 353 amino acid residues (residues 226 -578) are sufficient for the IP 3 binding with high affinity and specificity. Newton et al. (18) could delete the C terminus up to the residue 577. These results indicate that the critical region for the IP 3 binding should be localized between the residues 226 and 576. This critical IP 3 binding region of 351 amino acids shares 44% identity and 61% similarity with some interspersed diverse sequences in the corresponding region of other members of the IP 3 R family (Fig. 4). Within this region, only the alternative-splicing segment SI (15 amino acids, residues 318 -332) is unlikely to be requisite for the binding, since the IP 3 R1 SI(Ϫ) splicing variant, which lacks this region, retains binding affinity similar to that of the IP 3 R1 SI(ϩ) variant, which has this region (18). 2 On the other hand, a deletion of either 3 more N-terminal or 9 more C-terminal amino acids from this 351amino acid region completely abolished the binding activity. Mignery et al. (7,16) and Miyawaki et al. (17) have reported that any small deletions within this region cause a failure to bind IP 3 . Thus, residues 226 -576 were sufficient and close to the minimum portion required for specific IP 3 binding. Thus, we suggest that this portion forms the IP 3 binding core.
We mutated 41 basic amino acids (Arg and Lys) within the N-terminal 650-amino acid residues to the neutral amino acid, Gln, either singly or in pairs. Of these mutations, 10 single 2 F. Yoshikawa and T. Furuichi, unpublished data. Double and single dots indicate amino acids identical and functionally conserved, respectively, in all members of the IP 3 R family (mouse type 1 (6); rat types 1 (7), 2 (8), and 3 (9); human types 1 (11), 2 (12), and 3 (10,12); Xenopus (40); and Drosophila (41) IP 3 R) (middle lane) and of the RyR family (rabbit type 1 (42,43); human type 1 (43); and rabbit types 2 (44, 45) and 3 (46)) (lower lane). Conservative amino acid substitutions are defined as follows: Ser, Thr, Pro, Ala, and Gly; Asn, Asp, Glu, and Gln; His, Arg, and Lys; Met, Ile, Leu, and Val; Phe, Tyr, and Trp (47). The basic amino acids, Arg and Lys, are shown in bold. Of these, 41 basic residues were analyzed for specific IP 3 binding by site-directed mutagenesis to Gln. Ten single substitutions caused the reduction in the binding activity (Ç), and the rest showed no reduction in the binding activity (E). An alternative-splicing segment, SI (residues 318 -332), is underlined. The positions of the amino acid residues are shown on the left. substitutions of Gln for Arg-241, Lys-249, Arg-265, Arg-269, Arg-504, Arg-506, Lys-508, Arg-511, Arg-568, and Lys-569 markedly reduced the IP 3 binding activity. All 10 residues were identical among all IP 3 Rs, suggesting that they are functionally important. Three of them (Arg-265, Lys-508, and Arg-511) were critical, since the IP 3 binding was completely abolished, even by substitution with a single amino acid. The reduction in IP 3 binding activity caused by these site-directed substitutions is possibly due to a loss of the original side chain, which directly interacts with IP 3 , or to disruption of a local or global conformation for the binding. These 10 residues are scattered within the binding core, which is consistent with the results from the deletion studies, and can be classified into four segments (the first containing Arg-241 and Lys-249; the second, Arg-265 and Arg-269; the third, Arg-504, Arg-506, Lys-508, and Arg-511; and the fourth, Arg-568 and Lys-569). We suggest that on the tertiary structure of IP 3 R, these separated segments are positioned close to each other and form a positively charged pocket for binding to the negatively charged phosphate groups of IP 3 . The third segment is close to the residues 476 -501, which were labeled by photoaffinity ligand, suggesting that it is in the proximity of the ligand binding site (19). Even functionally conservative mutations between Arg and Lys (K508R, R511K, and K569R) markedly reduced the IP 3 binding activity like Gln or Ala substitutions, suggesting that the restricted basic amino acid residues in the higher order structure are requisite for the  specific interaction with IP 3 . In comparison with the wild-type T734, the mutant R568Q exhibited lower affinity for (1,4,5)IP 3 and a different binding specificity for various inositol phosphates, suggesting that Arg-568 is involved in not only high affinity binding with (1,4,5)IP 3 but also determination of binding specificity. Therefore, Arg-568 may be involved in recognition of the functional group at the equatorial position-1 of the inositol ring, since R568Q recognizes (4,5)IP 2 and (2,4,5)IP 3 with higher affinity but (1,4,5)IP 3 and (1,3,4,5)IP 4 with lower affinity than the wild type.
The Function of the N Terminus (Residues 1-225)-This study indicated that the first 225 amino acids are not requisite for the specific IP 3 binding, although there is significant sequence homology (64% identity and 76% similarity) in this region of the IP 3 R family. On the other hand, the deletion of only the first 31 amino acids resulted in a severe reduction in the binding activity. Such contradictory mutational effects were also found in serial N-terminal deletions up to residue 215. However, a deletion of 5 amino acids more toward the C terminus recovered the binding activity (D (1-220)). The binding activity was also recovered in the deletions as far as the residue 225 (D(1-223) and D(1-225)). These can be explained by two models. Serial deletions of the N terminus up to the residue 215 interfere with the higher order structure of the IP 3 binding site formed by the residues 226 -576, or the residues 216 -220 act as a part of the inhibitory determinant when deletions up to residue 215 are performed. Therefore, we tested whether the synthetic peptide (CNTSWKIVLFMK) corresponding to the residues 214 -225 inhibits the IP 3 binding of the cerebellar IP 3 R, T734, or the mutant D(1-223). However, as far as we tested, this peptide had no significant inhibitory effect (data not shown) although we could not rule out the possibility that it does not inhibit the binding in an intermolecular manner.
The binding affinity for IP 3 of the core region (residues 224 -579) was more than 10-fold higher than that of T734. This augmented affinity was probably due to deleting the N-terminal 223 amino acids from T734, since the mutant D(1-223) showed similar higher binding affinity (data not shown). Thus, we suggest that the N-terminal 225 amino acids are not directly responsible for, but may modulate, IP 3 binding (for example, the binding affinity).
Comparison of the N-terminal Portions between IP 3 R and the Ryanodine Receptor (RyR)-RyR is another intracellular Ca 2ϩ release channel originally identified in the sarcoplasmic reticulum of skeletal muscle or cardiac muscle. IP 3 R has fragmentary sequence homology with RyR in the N-terminal portion, suggesting involvement of this region in common receptorchannel function(s) (6,36). This seems to be supported by the fact that a single mutation of Arg-615 of the type 1 RyR (RyR1) to Cys causes porcine malignant hyperthermia (37). The porcine malignant hyperthermia RyR1 channels are hypersensitive to various modulators, suggesting that the region around Arg-615 is a regulatory domain of channel opening, or that it is involved in binding an unknown channel activator. This region of RyR1 is fragmentarily homologous with the corresponding region (residues 621-659) of IP 3 R1 (Fig. 4). Besides this region, there are four homologous fragments within the N-terminal 580 amino acids of IP 3 R1 (residues 115-197, 227-252, 276 -319, and 472-516) (6), of which three are within the IP 3 binding region defined in this study. Among 10 important basic residues for the IP 3 binding, three (Arg-241, Arg-504, and Arg-506) are conserved, but three critical residues (Arg-265, Lys-508, and Arg-511) are diverse in the RyR family (Fig. 4). Therefore, these homologous regions may be required for receptor-channel functions common in the intracellular Ca 2ϩ release channel superfamily, such as sensing activation signal(s), modulation, or the transduction of activating signal(s) to channel opening.
We defined the importance of basic amino acids for the binding of IP 3 R1 to IP 3 molecule, which is similar to that reported for some pleckstrin homology domains that bind IP 3 (27,28). However, the overall constitution of the binding site seems to be different from these, which is reflected in the binding affinity and specificity. On the other hand, IP 3 R may recognize the hydroxy groups and the inositol ring of IP 3 (20,38,39). The amino acids involved in these interactions in the critical region defined here require further analysis.