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Critical Regions for Activation Gating of the Inositol 1,4,5-Trisphosphate Receptor

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m300646200

ISSN

1083-351X

Autores

Keiko Uchida, Hiroshi Miyauchi, Teiichi Furuichi, Takayuki Michikawa, Katsuhiko Mikoshiba,

Tópico(s)

Receptor Mechanisms and Signaling

Resumo

To understand the molecular mechanism of ligand-induced gating of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+release channel, we analyzed the channel properties of deletion mutants retaining both the IP3-binding and channel-forming domains of IP3R1. Using intrinsically IP3R-deficient cells as the host cells for receptor expression, we determined that six of the mutants, those lacking residues 1–223, 651–1130, 1267–2110, 1845–2042, 1845–2216, and 2610–2748, did not exhibit any measurable Ca2+ release activity, whereas the mutants lacking residues 1131–1379 and 2736–2749 retained the activity. Limited trypsin digestion showed that not only the IP3-gated Ca2+-permeable mutants lacking residues 1131–1379 and 2736–2749, but also two nonfunctional mutants lacking residues 1–223 and 651–1130, retained the normal folding structure of at least the C-terminal channel-forming domain. These results indicate that two regions of IP3R1, viz. residues 1–223 and 651–1130, are critical for IP3-induced gating. We also identified a highly conserved cysteine residue at position 2613, which is located within the C-terminal tail, as being essential for channel opening. Based on these results, we propose a novel five-domain structure model in which both N-terminal and internal coupling domains transduce ligand-binding signals to the C-terminal tail, which acts as a gatekeeper that triggers opening of the activation gate of IP3R1 following IP3 binding. To understand the molecular mechanism of ligand-induced gating of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+release channel, we analyzed the channel properties of deletion mutants retaining both the IP3-binding and channel-forming domains of IP3R1. Using intrinsically IP3R-deficient cells as the host cells for receptor expression, we determined that six of the mutants, those lacking residues 1–223, 651–1130, 1267–2110, 1845–2042, 1845–2216, and 2610–2748, did not exhibit any measurable Ca2+ release activity, whereas the mutants lacking residues 1131–1379 and 2736–2749 retained the activity. Limited trypsin digestion showed that not only the IP3-gated Ca2+-permeable mutants lacking residues 1131–1379 and 2736–2749, but also two nonfunctional mutants lacking residues 1–223 and 651–1130, retained the normal folding structure of at least the C-terminal channel-forming domain. These results indicate that two regions of IP3R1, viz. residues 1–223 and 651–1130, are critical for IP3-induced gating. We also identified a highly conserved cysteine residue at position 2613, which is located within the C-terminal tail, as being essential for channel opening. Based on these results, we propose a novel five-domain structure model in which both N-terminal and internal coupling domains transduce ligand-binding signals to the C-terminal tail, which acts as a gatekeeper that triggers opening of the activation gate of IP3R1 following IP3 binding. inositol 1,4,5-trisphosphate IP3receptor IP3-induced Ca2+ release ryanodine receptor monoclonal antibody Inositol 1,4,5-trisphosphate (IP3)1 is a second messenger that is produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to activation by extracellular stimuli of the G protein- or tyrosine kinase-coupled receptors on the plasma membrane in various cell types (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6280) Google Scholar). IP3 mediates the release of Ca2+ from intracellular storage sites such as the endoplasmic reticulum by binding to the IP3 receptor (IP3R)/Ca2+ release channel. IP3-induced Ca2+ release (IICR) regulates numerous physiological processes, including fertilization, cell proliferation, development, muscle contraction, secretion, learning, and memory. In this signal transduction pathway, the IP3R works as a switch that converts the information carried by extracellular stimuli into intracellular Ca2+ signals. IP3-gated intracellular Ca2+ release channels are composed of four IP3R subunits (2Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Abstract Full Text PDF PubMed Google Scholar). There are at least three types of IP3Rs (IP3R1, IP3R2, and IP3R3) (3Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (181) Google Scholar), and they exist as both homo- and heterotetramers (4Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuichi T. Saruta T. Hasegawa M. Mikoshiba K. J. Biol. Chem. 1995; 270: 14700-14704Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The structure of IP3Rs has traditionally been divided into three functional domains (3Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (181) Google Scholar, 5Südhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (322) Google Scholar): the N-terminal ligand-binding domain; the modulatory/coupling domain; and the C-terminal transmembrane/channel-forming domain, which contains six putative membrane-spanning regions. The transmembrane region is required for the intermolecular interaction in the formation of a tetrameric complex (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar, 7Joseph S.K. Boehning D. Pierson S. Nicchitta C.V. J. Biol. Chem. 1997; 272: 1579-1588Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Sayers L.G. Miyawaki A. Muto A. Takeshita H. Yamamoto A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 323: 273-280Crossref PubMed Scopus (47) Google Scholar, 9Galvan D.L. Borrego-Diaz E. Perez P.J. Mignery G.A. J. Biol. Chem. 1999; 274: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and it is likely that the C-terminal cytoplasmic region just following the putative membrane-spanning regions has a supportive role in the association among the subunits (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar,9Galvan D.L. Borrego-Diaz E. Perez P.J. Mignery G.A. J. Biol. Chem. 1999; 274: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). An ion conduction pore has been proposed to be located in the hydrophobic segment between the fifth and sixth transmembrane regions (10Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Abstract Full Text PDF PubMed Google Scholar, 11Boehning D. Joseph S.K. Mak D.O. Foskett J.K. Biophys. J. 2001; 81: 117-124Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The primary sequence of the transmembrane domain adjacent to the pore-forming segment is highly homologous to that of the ryanodine receptor (RyR), another type of intracellular Ca2+ release channel, suggesting that these two channels might share a common structure for the conduction of Ca2+ ions. Each IP3R subunit has a single high affinity IP3-binding site (2Maeda N. Kawasaki T. Nakade S. Yokota N. Taguchi T. Kasai M. Mikoshiba K. J. Biol. Chem. 1991; 266: 1109-1116Abstract Full Text PDF PubMed Google Scholar). The IP3-binding core, a minimum essential region for specific IP3 binding (12Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), resides among residues 226–578 of mouse IP3R1 (2749 amino acids) (13Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (898) Google Scholar), and it contains 11 essential basic amino acids for IP3 binding (14Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. Michikawa T. Mikoshiba K. Ikura M. Nature. 2002; 420: 696-700Crossref PubMed Scopus (287) Google Scholar). The N-terminal 225 residues, which are close to the IP3-binding core, have been thought to function as a suppressor for IP3 binding because their deletion from the N-terminal 734-amino acid region results in significant enhancement of IP3-binding activity (12Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 15Yoshikawa F. Uchiyama T. Iwasaki H. Tomomori-Satoh C. Tanaka T. Furuichi T. Mikoshiba K. Biochem. Biophys. Res. Commun. 1999; 257: 792-797Crossref PubMed Scopus (42) Google Scholar). IICR is a positively cooperative process (16Meyer T. Wensel T. Stryer L. Biochemistry. 1990; 29: 32-37Crossref PubMed Scopus (159) Google Scholar, 17Hirota J. Michikawa T. Miyawaki A. Furuichi T. Okura I. Mikoshiba K. J. Biol. Chem. 1995; 270: 19046-19051Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), i.e. the binding of at least two IP3 molecules to a single tetrameric IP3R channel is required for channel opening. IP3 binding elicits a large conformational change in the N-terminal cytoplasmic portion of the IP3R (19Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (277) Google Scholar). Furthermore, the C-terminal cytoplasmic region following the transmembrane domain is thought to be involved in the IP3-induced gating of the receptor because monoclonal antibody (mAb) 18A10, whose epitope is located in the C-terminal portion of mouse IP3R1 (13Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (898) Google Scholar, 20Maeda N. Niinobe M. Nakahira K. Mikoshiba K. J. Neurochem. 1988; 51: 1724-1730Crossref PubMed Scopus (135) Google Scholar, 21Nakade S. Maeda N. Mikoshiba K. Biochem. J. 1991; 277: 125-131Crossref PubMed Scopus (86) Google Scholar), has an inhibitory effect on IICR, without causing any decrease in the affinity of the receptor for IP3 (21Nakade S. Maeda N. Mikoshiba K. Biochem. J. 1991; 277: 125-131Crossref PubMed Scopus (86) Google Scholar). Controlled trypsinization induces fragmentation of mouse IP3R1 into five major fragments, and all four N-terminal cytoplasmic fragments, which contain the IP3-binding core, are associated directly or indirectly with the remaining C-terminal fragment, which contains the channel domain (22Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The trypsinized IP3R retains significant IICR activity, indicating that intramolecular interaction within a subunit and/or intermolecular interaction between neighboring subunits could effect functional coupling between IP3 binding and channel opening (22Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). However, the sites of the interfaces between the cytoplasmic fragments and the channel domain and the molecular mechanism of their coupling remain to be elucidated. IICR has been shown to occur in a quantal manner in permeabilized cells and isolated endoplasmic reticulum membranes (23Muallem S. Pandol S.J. Beeker T.G. J. Biol. Chem. 1989; 264: 205-212Abstract Full Text PDF PubMed Google Scholar, 24Meyer T. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3841-3845Crossref PubMed Scopus (135) Google Scholar). The addition of submaximal concentrations of IP3 in the presence of Ca2+ pump inhibitors leads to the partial release of sequestered Ca2+, and the amount of released Ca2+ varies with the concentration of IP3 (24Meyer T. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3841-3845Crossref PubMed Scopus (135) Google Scholar). Although the Ca2+ release terminates abruptly, because it can be reinitiated by an additional increment in IP3concentration (24Meyer T. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3841-3845Crossref PubMed Scopus (135) Google Scholar), the rapid termination of Ca2+ release is not due to ordinary inactivation or desensitization of the receptor. Purified IP3Rs reconstituted into lipid vesicles reveal a quantal Ca2+ flux (17Hirota J. Michikawa T. Miyawaki A. Furuichi T. Okura I. Mikoshiba K. J. Biol. Chem. 1995; 270: 19046-19051Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Ferris C.D. Cameron A.M. Huganir R.L. Snyder S.H. Nature. 1992; 356: 350-352Crossref PubMed Scopus (112) Google Scholar), indicating that the quantal release of Ca2+ is an intrinsic property of the IP3R. Similar behavior was observed for the RyR, which mediates Ca2+-induced Ca2+ release from intracellular Ca2+ stores (26Györke S. Fill M. Science. 1993; 260: 807-809Crossref PubMed Scopus (277) Google Scholar), but has not been observed for other ligand-gated ion channels on the plasma membrane, suggesting that the quantal release is a fundamental and unique property of the intracellular Ca2+ release channels. To understand the molecular basis of the ligand-induced gating of the IP3R, we analyzed a series of internal deletion mutants and site-directed mutants of mouse IP3R1 expressed in intrinsically IP3R-deficient R23-11 cells (27Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (378) Google Scholar). We found that at least two regions and a cysteine residue are essential for IP3-dependent gating of IP3R1. These findings provide us with new insight into the gating mechanism of the IP3R. For transfection of mouse wild-type IP3R1 cDNA, pBact-STneoB-C1 (28Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (121) Google Scholar) was used. Seven deletion mutant cDNAs of mouse IP3R1, D651–1130, D1131–1379, D1267–2110, D1692–1731, D1845–2042, D1845–2216, and D2610–2748 (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar), were subcloned into pAneo (27Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (378) Google Scholar) at the SalI sites. To construct D1–223, an XhoI site was introduced at nucleotide 998 of mouse IP3R1 by PCR using green fluorescent protein-fused IP3R-D223 2Y. Tateishi, M. Hattori, T. Michikawa, M. Iwai, K. Uchida, T. Nakayama, T. Nakamura, T. Inoue, and K. Mikoshiba, unpublished data. as the template DNA. An XhoI-KpnI fragment isolated from green fluorescent protein-fused IP3R-D223 was ligated to a SalI-KpnI fragment of pBact-STneoB-C1. The resultant plasmid, pBact-STneoB-D1–223, uses nucleotides 998–1000, which correspond to an intrinsic methionine residue at position 224, as a start codon (ATG) for transcription. To construct D2736–2749, aKpnI-XhoI fragment isolated from enhanced green fluorescent protein-fused IP3R/Δ18A10 (29Zhang S. Mizutani A. Hisatsune C. Higo T. Bannai H. Nakayama T. Hattori M. Mikoshiba K. J. Biol. Chem. 2003; 278: 4048-4056Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) was ligated to a KpnI-SalI fragment of pBact-STneoB-C1. To substitute serine for cysteine at position 1976, 2610, or 2613 or at both positions 2610 and 2613 of mouse IP3R1, site-directed mutagenesis was performed with a MutanK kit (Takara) using primers containing the appropriate substitutions (5′-GTGGTTTTCAGACAGCAGCTG-3′ for nucleotides 6245–6265, 5′-CAGATGAAGCTCGTGGTTTTT-3′ for nucleotides 8146–8166, 5′-TTCCAAGCCGGAGATGAAGCA-3′ for nucleotides 8156–8176, and 5′-AAGCCGGAGATGAAGCTCGTGGT-3′ for nucleotides 8150–8172). The EcoRI fragment from the EcoRI site (nucleotide 6979) internal to the 3′-end of the mouse IP3R1 isolated from pBactS-C1 (13Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (898) Google Scholar) was subcloned into pBluescript SK(+). The BamHI fragment (2532 bp) of the mouse IP3R1 isolated from pBactS-C1 (13Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (898) Google Scholar) was subcloned into pUC118. These plasmids were used as template DNAs. After the mutatedEcoRI or BamHI fragments were put back into pBactS-C1, the mutated cDNAs were subcloned into pBact-STneoB (28Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (121) Google Scholar) at the SalI sites. All PCR products and mutations were confirmed by DNA sequencing. R23-11 cells (27Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (378) Google Scholar) were cultured in RPMI 1640 medium supplemented with 107 fetal calf serum, 17 chicken serum, 50 ॖm 2-mercaptoethanol, 4 mm glutamine, 100 units/ml penicillin, and 100 ॖg/ml streptomycin at 39.5 °C in 57 CO2. Expression plasmids were linearized and transfected into R23-11 cells by electroporation as previously described (27Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (378) Google Scholar) or by lipofection (Effectene, QIAGEN Inc.). 3H. Miyauchi, K. Uchida, T. Kirino, T. Michikawa, and K. Mikoshiba, manuscript in preparation. Several stable clones were selected in medium containing 2 mg/ml G418 (Sigma) ∼7–10 days after transfection. Expression of the IP3R and its mutants was confirmed by immunoblotting with mAbs 4C11 and/or 18A10 using cell lysates boiled in SDS-PAGE sample buffer (5 mm EDTA, 50 mm Tris-HCl, pH 6.8, 100 mm dithiothreitol, 27 SDS, and 107 glycerol). Immunoblot analysis was performed as described previously (22Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Membrane fractions were prepared in accordance with the protocol for mouse cerebella described by Michikawa et al. (18Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), with minor modifications. Cells were collected by centrifugation, washed twice with cold phosphate-buffered saline, and homogenized in ice-cold homogenization buffer (5 mm NaN3, 0.1 mm EGTA, 1 mm 2-mercaptoethanol, and 20 mm HEPES-NaOH, pH 7.4) containing protease inhibitors (0.1 mmphenylmethylsulfonyl fluoride, 10 ॖm leupeptin, 10 ॖm pepstatin A, and 10 ॖm E-64) by 40 strokes in a chilled glass-Teflon Potter homogenizer at 1000 rpm. The homogenate was centrifuged at 1100 × g for 10 min at 2 °C. The supernatant was centrifuged at 100,000 ×g in a Beckman TLA100.3 rotor for 30 min at 2 °C. The pellet was resuspended in an appropriate volume of wash buffer (600 mm KCl, 5 mm NaN3, 20 mm Na4P2O5, 1 mm 2-mercaptoethanol, and 10 mm HEPES-HCl, pH 7.2) containing protease inhibitors. The suspension was centrifuged at 1100 × g for 10 min, and the supernatant was centrifuged at 63,000 × g for 30 min at 2 °C. The pellet was finally suspended in an appropriate volume of Ca2+ release buffer (110 mm KCl, 10 mm NaCl, 5 mm KH2PO4, 1 mm 2-mercaptoethanol, and 50 mm HEPES-KOH, pH 7.2) containing protease inhibitors to a final concentration of ∼15 mg/ml protein. Ca2+ release buffer was passed over Chelex 100 (Bio-Rad) to eliminate any extra free Ca2+ before use. The membrane fractions were either used immediately or frozen in liquid nitrogen and stored at −80 °C until used. The IP3 binding assay was performed as described previously (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar). The membrane fractions (50–200 ॖg/tube) were incubated with 9.6 nm[3H]IP3 (PerkinElmer Life Sciences) in 100 ॖl of binding buffer (50 mm Tris-HCl, pH 8.0, 1 mm EGTA, and 1 mm 2-mercaptoethanol) for 10 min at 4 °C. After centrifugation, the pellets were dissolved in Solvable (PerkinElmer Life Sciences), and the radioactivities were measured with a scintillation counter (Beckman LS6500). Nonspecific binding was measured in the presence of 10 ॖm unlabeled IP3 (Dojindo Laboratories). The membrane fractions were suspended in Ca2+ release buffer supplemented with 1 ॖg/ml oligomycin (Sigma), 2 mm MgCl2, 25 ॖg/ml creatine kinase (Roche Applied Science), 10 mmcreatine phosphate (Sigma), and 2 ॖm Fura-2 (Molecular Probes, Inc.) and used at a concentration of 200–300 ॖg/ml protein. Fluorescence was recorded at 510 nm with alternate excitation of 340 and 380 nm (F340 andF380, respectively). Using a CAF-110 spectrofluorometer (Japan Spectroscopic Co.), signals were recorded every 0.01 s with MacLab Version 3.6 (ADInstruments) at 30 °C. When the Ca2+ uptake induced by the addition of 1 mm ATP reached a steady level, 2 ॖmthapsigargin was added to eliminate active Ca2+ uptake through intrinsic Ca2+ pumps. The rate of leakage from the membrane fractions following the addition of thapsigargin was almost linear. When the ratio of fluorescence intensity (F340/F380) reached 1.2, corresponding to ∼170 nm free Ca2+, various concentrations of IP3 were added. At the end of each experiment, 2 mm CaCl2 and 10 mmEGTA were added successively for normalization and calibration (30Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Limited trypsin digestion was performed as described previously (22Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Microsomal fractions (0.25–5 mg/ml) of wild-type and mutant IP3R1-expressing cells were incubated with 0.01–10 ॖg/ml trypsin in trypsinization buffer (120 mm KCl, 1 mm EDTA, 1 mm 2-mercaptoethanol, and 20 mm Tris-HCl, pH 8.0) at 35 °C for 10 min. The reaction was terminated by the addition of 50 ॖg/ml soybean trypsin inhibitor (Sigma) and 0.1 mm phenylmethylsulfonyl fluoride. After the addition of an equal volume of SDS-PAGE sample buffer, reaction mixtures were incubated at 55 °C for 30 min. The digested proteins were separated by 87 SDS-PAGE and then analyzed by Western blotting with anti-IP3R1 antibodies N1, 4C11, 10A6, 1ML1, and 18A10 and the anti-(1718–31) antibody (see Fig. 1A) (22Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). As previously reported (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar), we constructed 17 internal deletion mutants of mouse IP3R1. Among these mutants, we selected seven (Fig.1B) containing both the IP3-binding region (residues 226–578) and the putative transmembrane domain (residues 2276–2589) to investigate the critical regions for the coupling between ligand binding and channel opening. In addition, we constructed two mutants lacking residues 1–223 and 2736–2749, respectively (Fig. 1B). To express these mutant receptors, we introduced the mutant cDNAs into R23-11 cells and established stable cell lines by selection with 2 mg/ml G418. Fig.2A illustrates the results from Western blot analysis of the membrane fractions prepared from these stable cell lines using anti-IP3R1 polyclonal antibody 1ML1, whose epitope lies within residues 2504–2523 of IP3R1 (Fig. 1A) (10Michikawa T. Hamanaka H. Otsu H. Yamamoto A. Miyawaki A. Furuichi T. Tashiro Y. Mikoshiba K. J. Biol. Chem. 1994; 269: 9184-9189Abstract Full Text PDF PubMed Google Scholar). All of the mutant receptors except D2610–2748 were detected with an appropriate molecular mass (Fig. 2A). Because D2610–2748 was not well recognized by antibody 1ML1, we confirmed the expression of D2610–2748 by Western blot analysis using anti-IP3R1 mAb 4C11 (20Maeda N. Niinobe M. Nakahira K. Mikoshiba K. J. Neurochem. 1988; 51: 1724-1730Crossref PubMed Scopus (135) Google Scholar). As shown in Fig. 2B (open arrowhead), an additional signal with a low molecular mass was detected, indicating that degradation (or truncation) of D2610–2748 occurs in R23-11 cells. The IP3-binding activities of the internal deletion mutant receptors expressed in R23-11 cells were measured by equilibrium [3H]IP3 binding analysis as described previously (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar). There was no significant IP3-binding activity in the membrane fraction obtained from the R23-11 cells (data not shown). Therefore, we measured the ligand-binding activity of the exogenously expressed IP3R using membrane fractions obtained from the stable cell lines. The IP3-binding properties of wild-type and deletion mutant IP3Rs are summarized in TableI. Wild-type IP3R1 expressed in R23-11 cells showed a single high affinity IP3-binding site with a dissociation constant of 20 ± 5 nm(n = 3). This value is close to those of native IP3R1 expressed in the mouse cerebellum (31Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (234) Google Scholar) and cDNA-derived IP3R1 expressed in L cells (28Miyawaki A. Furuichi T. Maeda N. Mikoshiba K. Neuron. 1990; 5: 11-18Abstract Full Text PDF PubMed Scopus (121) Google Scholar), NG108-15 cells (6Miyawaki A. Furuichi T. Ryou Y. Yoshikawa S. Nakagawa T. Saitoh T. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4911-4915Crossref PubMed Scopus (135) Google Scholar), and Sf9 cells (32Yoneshima H. Miyawaki A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 322: 591-596Crossref PubMed Scopus (89) Google Scholar). Mutant receptors D1131–1379, D1692–1731, D2610–2748, and D2736–2749 exhibited binding affinity similar to that of wild-type IP3R1 (Table I). The IP3-binding affinity of mutant receptors D1267–2110, D1845–2042, and D1845–2216 was 2–3-fold lower (Table I), and mutant D651–1130 had 7.5-fold lower affinity for IP3 (Table I). Mutant D1–223 exhibited, however, significantly higher affinity for IP3 (Table I), consistent with a previous report showing that residues 1–223 act as a suppressor for IP3 binding (12Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). It has been reported that IP3 binding to the IP3R is not cooperative (31Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (234) Google Scholar, 33Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Abstract Full Text PDF PubMed Google Scholar), and the same property holds true for the wild-type receptor and all of the mutant receptors except D1–223 expressed in R23-11 cells (Table I). Both the Western blot (Fig. 2) and IP3 binding (Table I) analyses showed that the amount of IP3R protein expressed in each cell line was different. The amounts of the mutant IP3Rs expressed were in the range of 1.5–9.2 pmol/mg of protein (Table I); and therefore, we used two stable cell lines (KMN13 and KMN107)2 expressing different amounts of wild-type IP3R1 as controls in the following experiments. TheBmax values for KMN13 and KMN107 were 13 ± 9 and 0.77 ± 0.2 pmol/mg of protein, respectively (Table I).Table IIP3-binding properties of the deletion mutantsRecombinant proteinAffinity (Kd)BmaxHill coefficientnmpmol/mg proteinWild-type KMN1320 ± 513 ± 91.0 ± 0.05 KMN10777 ± 500.77 ± 0.20.97 ± 0.02D1-2231.5 ± 0.41-ap < 0.05 by Student's ttest.14 ± 31.5 ± 0.21-bp < 0.01 by Student's t test.D651-1130150 ± 301-ap < 0.05 by Student's ttest.5.2 ± 30.85 ± 0.1D1131-137912 ± 19.2 ± 71.1 ± 0.2D1267-211057 ± 51-ap < 0.05 by Student's ttest.4.2 ± 11.0 ± 0.04D1692-173122 ± 63.8 ± 20.99 ± 0.05D1845-204241 ± 31-ap < 0.05 by Student's ttest.1.7 ± 0.91.0 ± 0.08D1845-221648 ± 101-ap < 0.05 by Student's ttest.2.1 ± 0.50.92 ± 0.02D2610-274867 ± 501.5 ± 11.0 ± 0.07D2736-274923 ± 57.2 ± 0.40.95 ± 0.07Values are expressed as means ± S.D. (n = 3).1-a p < 0.05 by Student's ttest.1-b p < 0.01 by Student's t test. Open table in a new tab Values are expressed as means ± S.D. (n = 3). To investigate the Ca2+ release activity of the mutant IP3Rs, IICR from the membrane fractions prepared from each stable cell line was measured in the presence of the Ca2+ pump inhibitor thapsigargin. No Ca2+ release was observed from membrane fractions prepared from R23-11 cells even after the addition of 10 ॖmIP3 (Fig. 3A), indicating that using R23-11 cells as host cells for transfection allows evaluation of definite Ca2+ release activity by exogenously expressed IP3Rs. Fig. 3B shows the time course of the Ca2+ release mediated by recombinant wild-type IP3R1 after the addition of various concentrations of IP3. Both the rate and amplitude of Ca2+ release depended on the concentration of IP3 added, indicating that IP3R1 expressed in R23-11 cells exhibits the quantal Ca2+ release that is known to be an intrinsic property of native IP3R1 (17Hirota J. Michikawa T. Miyawaki A. Furuichi T. Okura I. Mikoshiba K. J. Biol. Chem. 1995; 270: 19046-19051Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Ferris C.D. Cameron A.M. Huganir R.L. Snyder S.H. Nature. 1992; 356: 350-352Crossref PubMed Scopus (112) Google Scholar). As previously reported (11Boehning D. Joseph S.K. Mak D.O. Foskett J.K. Biophys. J. 2001; 81:

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