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J. Biol. Chem., Vol. 277, Issue 24, 21115-21118, June 14, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21115    most recent C200244200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamada, K. Articles by Mikoshiba, K. Search for Related Content PubMed PubMed Citation Articles by Hamada, K. Articles by Mikoshiba, K. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Two-state Conformational Changes in Inositol 1,4,5-Trisphosphate Receptor Regulated by Calcium^*

Kozo Hamada^§, Tomoko Miyata^¶, Kouta Mayanagi^¶, Junji Hirota^**, and Katsuhiko Mikoshiba

From the  Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan, the ^¶ Biomolecular Engineering Research Institute (BERI), 6-2-3 Furuedai, Suita, Osaka, 565-0874, Japan, the  Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan, and the  Calcium Oscillation Project, International Cooperative Research Project (ICORP), Japan Science and Technology Corporation (JST), 3-14-4, Shirokanedai, Minato-ku, Tokyo 108-0071, Japan

Received for publication, April 23, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Inositol 1,4,5-trisphosphate receptor (IP₃R) is a highly controlled calcium (Ca²⁺) channel gated by inositol 1,4,5-trisphosphate (IP₃). Multiple regulators modulate IP₃-triggered pore opening by binding to discrete allosteric sites within IP₃R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca²⁺, the most pivotal regulator, induced marked structural changes in the tetrameric IP₃R purified from mouse cerebella. Electron microscopy of the IP₃R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca²⁺ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP₃-binding domains, assigned by binding to heparin-gold. Ca²⁺-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca²⁺ appeared to regulate IP₃ gating activity through the rearrangement of functional domains.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Inositol 1,4,5-trisphosphate receptor (IP₃R)¹ is a tetrameric ion channel that release Ca²⁺ from intracellular stores in response to the binding of 1,4,5-trisphosphate (IP₃), a second messenger generated by various extracellular stimuli, neurotransmitters, neuromodulators, hormones, and lights (1, 2). The IP₃R is widely distributed in living systems and plays pivotal roles in fundamental processes including fertilization, cellular proliferation and differentiation, cellular signaling, and vesicle secretion (2). Molecular cloning studies have revealed that there are three isoforms of IP₃R and that alternative splicing results in several variants of the IP₃R (2). These divergent primary structures of the IP₃R and their differential distributions have been assumed to award the functional diversity of IP₃R by nature.

The most characterized type 1 IP₃R (IP₃R1), a predominant type in rodent cerebellar endoplasmic reticulum (ER) and spine apparatus, plays an integral role in Ca²⁺ signaling (3-5) and neural plasticity (6, 7). The protomer of IP₃R1, a 2749-amino acid polypeptide (M_r 313,000), contains the IP₃-binding core (residues 226-578), membrane-spanning domains (residues 2276-2589), and widespread allosteric sites for intracellular effector molecules (Ca²⁺, calmodulin, and ATP) and for phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, Ca²⁺/calmodulin-dependent protein kinase II, and tyrosine kinase) (2). These cumulative allosteric regulations imply a structural paradigm for global conformational changes within the higher ordered structure of IP₃R1.

Because Ca²⁺ rigorously determines the channel activity of IP₃R and Ca²⁺-dependent behavior of IP₃R is considered to be crucial for spatiotemporal organizations of Ca²⁺ signaling (1, 4), the most important regulator for IP₃R is Ca²⁺ per se. Previous functional analysis indicates that a low Ca²⁺ level acts as an essential coagonist for IP₃-gated Ca²⁺ release and a high Ca²⁺ level inversely acts as a feedback repressor (4, 8, 9) via Ca²⁺/calmodulin in part (10). Thereby we assumed that Ca²⁺ could induce alterations in conformational states of IP₃R1 underlying such dynamic regulations. An investigation of this hypothesis requires information about structural rearrangements that has heretofore been unclear because of the structural polymorphism within IP₃R particles, which is partially due to their fragile architectures, presented by previous electron microscopic studies (11-14). To address this issue, we improved rapid purification of the IP₃R1 channel so that we could use electron microscopic study to visualize the domain arrangement and to investigate its structural change by Ca²⁺.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Purification of IP₃R1-- Immunoaffinity purification of IP₃R1 was performed as described previously (15) with the following modifications. Microsomal membrane (3 mg/ml), prepared from mouse cerebella, was solubilized in 50 mM Tris buffer (pH 7.5) containing 1% (w/v) CHAPS, 150 mM KCl, 2 mM dithiothreitol, 200 µM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, 10 µM leupeptin, 10 µM E-64, and 0.2 mM CaCl₂ or 1 mM EDTA. The solubilized IP₃R1 was mixed with pep6Ab-immobilized beads, incubated at 4 °C, washed, and eluted with 20 µM pep 6. The [³H]-IP₃ binding assay was performed as described previously (11).

Electron Microscopy-- The purified IP₃R1 (0.5 µl) was injected into 9.5 µl of 50 mM Tris buffer (pH 7.5) containing 1 mM CaCl₂, 1 mM EDTA, or 1 mM EGTA and incubated for 30 min on ice. For heparin-gold labeling, purified IP₃R1 was mixed with a 5-10-fold molar excess of heparin-gold (Sigma) and incubated for 10 min on ice. An aliquot of the mixture (4 µl) was applied onto carbon-coated copper grids. Excess solution was removed by filter paper, and the IP₃R1 particles were stained with 2 µl of 1% (w/v) uranyl acetate solution. Dried grids were examined on a JEM 1200 EX transmission electron microscope (JEOL) operated at 80-kV acceleration voltage. Micrographs were taken at magnifications ranging from ×25,000 to ×50,000.

Partial Proteolysis-- Partial proteolysis experiments were carried out in the solution containing IP₃R1 and lysyl endopeptidase (Lys-C). Reaction mixtures were incubated for 30 min at 37 °C. The proteolysis was stopped by heat treatments at 55 °C for 15 min in the sample buffer including SDS. Proteolytic fragments were analyzed by discontinuous PAGE (5 or 10% gel) and immunoblotting using monoclonal antibodies 18A10 and 4C11 (11, 15).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We purified IP₃R1 to apparent homogeneity as judged by gel electrophoresis (Fig. 1A) from mouse cerebella, which was functionally active as estimated by the specific activity of maximum binding to IP₃ (3 nmol/mg of protein). The purified IP₃R1 was negatively stained and imaged in a transmission electron microscope. Electron microscopy explicitly showed two distinct structures in negatively stained samples. One was a windmill-like structure (Fig. 1B), and another was a square-shaped structure (Fig. 1C). The windmill structure contained four segregated radial wings and a central core. Each wing structure appeared to be composed of two domains: a globular domain, which often exhibited a central spot that was densely stained, and a constricted segment forming a bridge between the globular domain and the central core domain. The dimension of the windmill structure was 31 ± 2 nm (n = 76) from the tip of one wing to the tip of the opposite wing. The globular domain in the wing was 8.1 ± 0.8 nm (n = 30) in diameter, and the central core was 9.8 ± 0.6 nm (n = 15) in diameter. The shape and dimensions of the windmill structure are consistent with those previously reported for IP₃R isolated from smooth muscle (12). The dimensions of the square structure were 19 ± 1 nm (n = 54) on a side and 24 ± 1 nm (n = 54) on a diagonal line (Fig. 1C), similar to that of small dense projections in the smooth ER of rat Purkinje cells (14). Comparison with the dimensions of ryanodine receptor, another intracellular Ca²⁺ channel (16), provides support that the projected size of the square structure in this study is reasonable because of the ratio in the molecular mass of the protomer. We also found that the IP₃R1 particles appeared as other forms, suggesting their variances of orientation or intrinsic flexibilities.

View larger version (64K): [in this window] [in a new window]   Fig. 1.   Dual discrete structures of IP3R1. A, purified IP3R (1.2 µg) on SDS-PAGE (5% gel) stained with Coomassie Brilliant Blue. Electron microscopic images of negatively stained IP3R1 show a typical top view of a windmill-shaped particle (B) and a typical top view of a square-shaped particle (C). An arrow in B indicates a globular domain at the tip of a radial wing. The scale bar represents 20 nm.

Our microscopic data revealed two distinct states of the IP₃R1 molecule, leading us to the hypothesis that the conformation of IP₃R1 alters. We tried to capture a Ca²⁺-dependent transition between the dual structures by imaging IP₃R1 particles injected into 1 mM CaCl₂, 1 mM EDTA, or 1 mM EGTA. Significantly the windmill structures were abundantly observed in the presence of Ca²⁺ (Fig. 2A). In contrast, the relative abundance of windmill structures appeared to decrease in specimens prepared in solution containing 1 mM EDTA (Fig. 2B). For statistical evaluation, we counted the windmill particles with more than three identifiable wings and the square forms with a homologous dimension, which had no wing, on electron micrographs. Quantitative analysis clearly indicates a significant difference in the ratio of the two structures in a Ca²⁺-dependent manner (Fig. 2C). Readdition of CaCl₂ into the IP₃R1 purified with EDTA restored the windmill configuration, and the number of windmill structures showed a marked reduction upon readdition of EDTA into the IP₃R1 purified in the presence of Ca²⁺, indicating that the structural rearrangements are reversible (Fig. 2C). IP₃ did not induce significant changes in each state at this resolution, thus the binding of IP₃ may cause a fine structural change to open the channel.

View larger version (88K): [in this window] [in a new window]   Fig. 2.   Structural changes in the IP3R1 particle. Electron micrographs of IP3R1 injected into the solution containing 1 mM CaCl2 (A) or 1 mM EDTA (B). Marked projections respectively show top or tilted views of windmill particles (circled) and square particles (boxed). Bars, 100 nm. C, relative abundance of windmill and square particles. IP3R1 was purified in 0.2 mM CaCl2 (a), 1 mM EDTA (b), or neither CaCl2 nor EDTA (c), and then it was injected into 1 mM CaCl2, 1 mM EGTA, or 1 mM EDTA. Ratios were calculated from the identifiable windmill and square structures counted on electron micrographs. For statistical evaluations on each condition, 5-11 fields were evaluated resulting in a total of 169-685 molecules indicated at the top of each bar. The standard deviation was calculated by comparing different micrographs.

Our findings provide the first evidence of structural alterations in IP₃R1 molecules. The structural alteration could account for the polymorphism in IP₃R particles presented by previous electron microscopic studies (11-14). The unique architectures and conspicuous conformational changes within the IP₃R1 particle differ remarkably from those in the ryanodine receptor particle (17, 18). These differences might result from intrinsic properties of the gating machinery of IP₃R1.

To correlate the structural changes in IP₃R1 observed by electron microscopy with changes in solution, we monitored its sensitivity to limited protease digestion. The patterns of degradative intermediates were clearly Ca²⁺-dependent (Fig. 3). In particular, a 38-kDa fragment detected with 4C11 within the IP₃-binding domain was markedly generated by cleavage in CaCl₂ solution; however, a 48-kDa intermediate and a 38-60-kDa ladder of bands detected with 4C11 were dominantly observed in EDTA solution (Fig. 3B). Furthermore a C-terminal 130-kDa fragment detected with 18A10 was abundantly detected by cleavage of purified IP₃R1 in CaCl₂ solution compared with 85- and 120-kDa fragments (Fig. 3B). These results suggest structural changes in purified IP₃R1 rather than a simple acceleration or regulation of proteolysis by Ca²⁺. The degradation by contaminant protease, such as Ca²⁺-activated calpains, was insignificant because of negligible production of digested proteins without Lys-C (Fig. 3). In addition, we also investigated the dynamic property of IP₃R1 in crude microsomal membrane. The 38- and 30-kDa fragments detected with 4C11 were evenly precipitated in both CaCl₂ and EDTA solutions; however, these fragments were more releasable to supernatant fractions in the presence of CaCl₂ than in EDTA (Fig. 3C). We also confirmed the reproducibility of these proteolysis experiments by using 0.5 mM EGTA and 0.2 mM CaCl₂ solution. This heightened ability to release may be related to the structural changes within IP₃R1 embedded in the microsomal membrane. Taken together, these biochemical data strongly support the presence of structural alterations in IP₃R1.

View larger version (63K): [in this window] [in a new window]   Fig. 3.   Effects of Ca2+ on partial proteolysis. A, fragments (arrowheads) of purified IP3R digested by Lys-C in the presence of 1 mM CaCl2. The intact IP3R is marked by an asterisk. The products were probed with 18A10. B, fragments of purified IP3R1 digested in the presence of 2.5 mM EDTA (left four lanes) or 1 mM CaCl2 (right four lanes). The products were probed with 4C11 (upper) and 18A10 (lower). An asterisk indicates a ladder of bands (38-60 kDa). C, release of digested fragments from crude microsome membrane. The gels show fragments in 15,000 × g supernatants (sup) and precipitates (ppt). The products were detected by 4C11. Microsome fractions were pretreated with 1 mM EDTA (left) or 1 mM CaCl2 (right) and digested by Lys-C (0-10 µg/ml) at 4 °C (open bars) or 37 °C (solid bars). D, schematic representation of Ca2+-dependent fragments (solid bars). The middle row indicates boundaries of partially digested fragments shown in A. Binding sites for antibodies and heparin, indicated by dotted lines, and IP3-binding and channel regions (bottom) are based on previous studies (2, 20, 21).

To determine how functional domains are arranged, we used colloidal gold conjugated with heparin, which is a competitive inhibitor of IP₃ binding (19) and specifically binds to the N-terminal IP₃-binding region (20). Heparin-gold particles bound not only to the windmill structure but also to the square form (Fig. 4, A and B). In the windmill structure, the gold particles bound to the globular domain of the radial wings (Fig. 4A). In the square form, the gold particles attached at the sites close to the corners (Fig. 4B). We assigned these heparin-binding domains to the N-terminal IP₃-binding domain. Our results provide the first evidence of domain arrangement in quaternary configurations of the IP₃R1 particle. In both states, the distribution of peripheral IP₃-binding domains occurred away from the center, a plausible Ca²⁺ gateway, by over 5 nm, suggesting that a long range allosteric transmission took place underlying the IP₃-gated Ca²⁺ release.

View larger version (114K): [in this window] [in a new window]   Fig. 4.   Mapping of heparin-binding sites and proposed flapping model for structural rearrangements within IP3R1. A and B, typical top view images (upper rows) of windmill (A) and square (B) particles and those bound to heparin-gold (middle rows). The contours are shown below each particle to delineate the heparin-gold (closed circles) and the IP3R (dotted lines). The scale bar represents 20 nm. C, IP3-binding domain (light gray), channel domain (gray), and bridge domain (white). Black arrows indicate plausible domain movements.

The comparison between dual structures and mapping of heparin-binding sites indicates that the structural transition from square to windmill is caused by the relocation of functional domains. Therefore, we propose a "flapping model" for the large scale rearrangements in IP₃R1 (Fig. 4C). The windmill structure may be a consequence of the IP₃-binding domain splitting from the channel domain. The dynamic flapping may be mediated by the bridge domain, which may act as a hinge structure. Additionally the digested IP₃R retains the assembly of domains under Ca²⁺-free conditions (21, 22); thus interdomain coupling may also stabilize the more compact square structure. The Ca²⁺-dependent cleavage sites and releasable regions presented here are candidates for the apparent hinge and interface structures linking between functional domains.

Our findings show that the functional regulator altered the relative locations of IP₃-binding and channel domains even if there is no IP₃, further emphasizing that the domain arrangement is crucial for the transmission of IP₃ binding cues to Ca²⁺ pores. Two discrete configurations of IP₃R1 imply dual relay modes controlling the allosteric transmission. Which of two structures is more active? As it appears to have an advantage for direct transferring of conformational changes by IP₃ binding toward the central channel, we cannot exclude the possibility that the compact square form is an active state. Based on single channel analysis, however, high Ca²⁺ only acts as an activator toward purified IP₃R1 (10). Hence it is favorable that the windmill structure is more active. In this case, it is conceivable that the bridge domain presents an effective relay of ligand binding signals. This notion fascinates us as a new type of mechanical control on ligand gating behavior. The three-dimensional structure at much higher resolution will answer our central question on the precise pathway of allosteric transmission from the IP₃-binding core to the Ca²⁺ gateway underlying IP₃-gated channel opening.

Ca²⁺ dependence of IP₃R is known to interplay with the allosteric regulation by ATP (23) and the cooperative gating by IP₃ (24). Thereby the Ca²⁺-dependent global conformational changes may concern primary states for other allosteric ligands. Since IP₃R is known to interact with phosphatidylinositol 4,5-bisphosphate incorporated in the plasma membrane (25), with the transient receptor potential protein, which is a calcium channel assumed to be involved in capacitative Ca²⁺ entry (26), and with the Homer protein linking with metabotropic glutamate receptor involved in neural plasticity (27), it is interesting to study how the dramatic conformational change of IP₃R1 alters the association with these signaling molecules. Our novel model for structural rearrangements and the methodology presented here should be useful for understanding of the further biological significance of structural plasticity within IP₃R1 in neural systems.

	ACKNOWLEDGEMENTS

We thank A. Terauchi (JST) for excellent technical assistance; Y. Kimura (BERI) and H. Sagara (Tokyo University) for discussion about electron microscopy; A. Mizutani (RIKEN) for support in protein purification and for biochemical advice; S. Ohmi (Tokyo University) for peptide synthesis; and A. Takahashi (RIKEN) for 18A10 and 4C11.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 81-3-5449-5320; Fax: 81-3-5449-5420; E-mail: hamada@ims.u-tokyo.ac.jp.

^** Present address: Laboratory of Vertebrate Developmental Neurogenetics, The Rockefeller University, 1230 York Ave., New York, NY 10021.

Published, JBC Papers in Press, April 29, 2002, DOI 10.1074/jbc.C200244200

	ABBREVIATIONS

The abbreviations used are: IP₃R, inositol 1,4,5-trisphosphate receptor; IP₃, inositol 1,4,5-trisphosphate; IP₃R1, type 1 IP₃R; ER, endoplasmic reticulum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Lys-C, lysyl endopeptidase.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/24/21115    most recent C200244200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hamada, K. Articles by Mikoshiba, K. Search for Related Content PubMed PubMed Citation Articles by Hamada, K. Articles by Mikoshiba, K. Social Bookmarking                      What's this?