Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

Katayama, Eisaku; Funahashi, H; Michikawa, Takayuki; Shiraishi, T; Ikemoto, Takaaki; Iino, Masashi; Mikoshiba, Katsuhiko

doi:10.1002/j.1460-2075.1996.tb00865.x

Cited by 67 publications

(56 citation statements)

References 49 publications

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“…The structural alteration could account for the polymorphism in IP 3 R particles presented by previous electron microscopic studies (11)(12)(13)(14). The unique architectures and conspicuous conformational changes within the IP 3 R1 particle differ remarkably from those in the ryanodine receptor particle (17,18).…”

Section: Resultsmentioning

confidence: 82%

“…Thereby we assumed that Ca 2ϩ could induce alterations in conformational states of IP 3 R1 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 IP 3 R particles, which is partially due to their fragile architectures, presented by previous electron microscopic studies (11)(12)(13)(14). To address this issue, we improved rapid purification of the IP 3 R1 channel so that we could use electron microscopic study to visualize the domain arrangement and to investigate its structural change by Ca 2ϩ .…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Hamada¹,

Miyata²,

Mayanagi³

et al. 2002

Journal of Biological Chemistry

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Inositol 1,4,5-trisphosphate receptor (IP 3 R) is a highly controlled calcium (Ca 2؉ ) channel gated by inositol 1,4,5-trisphosphate (IP 3 ). Multiple regulators modulate IP 3 -triggered pore opening by binding to discrete allosteric sites within IP 3 R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca 2؉ , the most pivotal regulator, induced marked structural changes in the tetrameric IP 3 R purified from mouse cerebella. Electron microscopy of the IP 3 R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca 2؉ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP 3 -binding domains, assigned by binding to heparin-gold. Ca 2؉ -dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca 2؉ appeared to regulate IP 3 gating activity through the rearrangement of functional domains.Inositol 1,4,5-trisphosphate receptor (IP 3 R) 1 is a tetrameric ion channel that release Ca 2ϩ from intracellular stores in response to the binding of 1,4,5-trisphosphate (IP 3 ), a second messenger generated by various extracellular stimuli, neurotransmitters, neuromodulators, hormones, and lights (1, 2). The IP 3 R 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 IP 3 R and that alternative splicing results in several variants of the IP 3 R (2). These divergent primary structures of the IP 3 R and their differential distributions have been assumed to award the functional diversity of IP 3 R by nature.The most characterized type 1 IP 3 R (IP 3 R1), a predominant type in rodent cerebellar endoplasmic reticulum (ER) and spine apparatus, plays an integral role in Ca 2ϩ signaling (3-5) and neural plasticity (6, 7). The protomer of IP 3 R1, a 2749-amino acid polypeptide (M r 313,000), contains the IP 3 -binding core (residues 226 -578), membrane-spanning domains (residues 2276 -2589), and widespread allosteric sites for intracellular effector molecules (Ca 2ϩ , calmodulin, and ATP) and for phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, Ca 2ϩ / 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 IP 3 R1.Because Ca 2ϩ rigorously determines the channel activity of IP 3 R and Ca 2ϩ -dependent behavior of IP 3 R is considered to be crucial for spatiotemporal organizations of Ca 2ϩ signaling (1, 4), the most important regulator for IP 3 R is Ca 2ϩ per se. Previous functional analysis indicates that a low Ca 2ϩ level acts a...

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Section: Resultsmentioning

confidence: 82%

mentioning

confidence: 99%

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

Hamada¹,

Miyata²,

Mayanagi³

et al. 2002

Journal of Biological Chemistry

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“…As can be seen in Figure 5, when the rate constants for each phase are plotted, the calculated Hill coefficients for the two phases appear to be identical and non-co-operative (h l 1.0). Although the receptor, in its native state, is believed to exist as a tetramer [25,26], these findings show that IICR is non-cooperative. This would suggest that there is no beneficial interaction between receptor monomers in influencing channel gating, and argues for a simple mechanism relating InsP $ binding to Ca# + release.…”

Section: Discussionmentioning

confidence: 99%

“…It is widely believed from electron microscopy evidence [26] that one functional channel exists per InsP $ receptor tetramer. To reconcile the presence of a single channel per tetramer with the non-co-operativity of Ca# + release, elaborate models (other than those previously proposed, which have assumed some co-operativity [27]) need to be developed.…”

Section: Discussionmentioning

confidence: 99%

Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes

Mezna

Michelangeli

1997

Biochemical Journal

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Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca# + concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP $ ) receptors. Thimerosal can have both sensitizing (at concentrations of 2 µM) and inhibitory (at concentrations of 2 µM) effects on InsP $ -induced Ca# + release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a biexponential process with a fast and a slow component. At a maximal InsP $ concentration (20 µM), the fast phase had a rate constant of 0.9 s −" and the slow phase had a rate constant of 0.4 s −" . The amplitudes of the two phases were 60 % and 40 % respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-

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“…Chadwick et al (29) described a ''pinwheel''-like structure with fourfold symmetry, 25 ϫ 25 nm in size. Katayama et al (30) described a native 2D lattice on the surface of the intracellular membrane of freeze-etched and platinum-shadowed Purkinje neurons, measuring Ϸ14 ϫ 16 nm. Packing considerations suggest that our IP 3 R 3D structure may be accommodated in such an array, particularly if potential flexibility in the petal subdomain is taken into account.…”

Section: Comparison With Previous Structural Data On the Ip3rmentioning

confidence: 99%

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Fonseca¹,

Morris²,

Nerou³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Materials and Methods Purification of IP3R.A membrane fraction was prepared from rat cerebella (17), resuspended (50 mg͞ml in 50 mM Tris͞1 mM EDTA, pH 8.3), and solubilized by the addition of 1% Triton X-100. After centrifugation (100,000 ϫ g for 60 min), the supernatant, supplemented with 250 mM NaCl, was loaded onto an Econopac heparin column (1 ml, Bio-Rad), washed with 25 ml of medium A (50 mM Tris͞250 mM NaCl͞10% glycerol, pH 8.3), supplemented with 1 mM EDTA, 1% Surfact-Amps X-100 (Pierce), and then with medium A supplemented with 0.1% Surfact-Amps X-100 (50 ml). IP 3 R was eluted from the heparin column directly onto a 2-ml Con A-agarose column (Sigma) by using medium A supplemented with 0.1% Surfact-Amps X-100 and 50 M decavanadate (18). After washing with 150 ml of medium B (50 mM Tris͞100 mM NaCl͞10% glycerol͞0.1% Surfact-Amps X-100, pH 8.3), IP 3 R was eluted by overnight incubation with 4 ml of medium B containing 800 mM methyl mannopyranoside (17). To assess the quaternary structure of the purified receptor, 0.2-ml fractions of the final eluate were loaded onto linear 10-30% sucrose gradients (10 ml) in 50 mM Tris͞1 mM EDTA͞0.1% Surfact-Amps X-100, pH 8.3, and centrifuged (134,000 ϫ g for 60 min). Gradients were calibrated by using a high-molecular-weight gel filtration standards kit (Amersham Biosciences). Size-exclusion HPLC was preformed by using a Zorbax GF-450 column (Jones Chromatography, Lakewood, CO) coupled to a Kontron system (Kontron Instruments, Watford, U.K.). All media included the following mixture of protease inhibitors: 150 nM aprotinin, 1 M pepstatin, 20 g͞ml soybean trypsin inhibitor, 1 mM benzamidine, 250 M PMSF, 250 M captopril, and 1 M bestatin.

show abstract

Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

Cited by 67 publications

References 49 publications

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

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

Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

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