Airway pathology in severe asthma is related to airflow obstruction but not symptom control.

BACKGROUND: Patients with asthma present structural and inflammatory alterations that are believed to play a role in disease severity. However, airway remodeling and inflammation have not been extensively investigated in relation to both symptom control and airflow obstruction in severe asthmatics. We aimed to investigate several inflammatory and structural pathological features in bronchial biopsies of severe asthmatics that could be related to symptom control and airflow obstruction after standardized treatment. METHODS: Fifty severe asthmatics received prednisone 40 mg/d for 2 weeks and maintenance therapy with budesonide/formoterol 400/12 µg twice daily + budesonide/formoterol 200/6 µg as needed for 12 weeks. Endobronchial biopsies were performed at the end of 12 weeks. We performed extensive immunopathological analyses of airway tissue inflammation and remodeling features in patients stratified by asthma symptom control and by airflow obstruction. RESULTS: Airway tissue inflammation and remodeling were not associated with symptom control. Asthmatics with persistent airflow obstruction had greater airway smooth muscle (Asm) area with decreased periostin and transforming growth factor beta-positive cells within Asm bundles, in addition to lower numbers of chymase-positive mast cells in the submucosa compared to patients with nonpersistent obstruction. CONCLUSIONS: Symptom control in severe asthmatics was not associated with airway tissue inflammation and remodeling, although persistent airflow obstruction in these patients was associated with bronchial inflammation and airway structural changes.

p53 contains a DNA break-binding motif similar to the functional part of BRCT-related region of Rb.

The BRCT regions are two repeating structures at BRCA1 carboxyl-terminus and are ubiquitous in some proteins involved in DNA repair and cell cycle checkpoints. It was shown that BRCTs of TopBP1, BRCA1, and BRCT-Ws of Rb bound DNA ends and breaks. We indicate here that the C-terminus of p53 tumor suppressor contains a DNA binding motif (residues 327-333 in human), whose features are similar to those of the part of BRCT-W in Rb with DNA binding activity. The short motif was required for the gel retardation activity of DNA fragments, since residues 311-333 showed the activity while residues 311-326 showed no activity. Significant numbers of total p53 and its fragments with the motif formed multimerizing complexes and associated with DNA ends and breaks. These results suggest the common presence of DNA binding motifs that can recognize DNA ends or damages in major tumor suppressors, Rb, BRCA1 and p53. The oncogenic activity of p53 C-terminus (residues 311-393) required both the DNA damage recognition motif and the repair enzyme-associating domain.

The BRCT regions of tumor suppressor BRCA1 and of XRCC1 show DNA end binding activity with a multimerizing feature.

The BRCT regions are two repeating structures in BRCA1 at the carboxyl-terminus and are ubiquitous in some proteins involved in cell cycle checkpoint and in DNA repair. Here, using electron microscopy, we show direct evidence that the BRCT regions of BRCA1 bound double-strand breaks of DNA. The BRCT regions could multimerize thus forming large protein particles. Smeared patterns of DNA fragments were consistently shown in the gel retardation assay. A single BRCT was sufficient for DNA binding. The smeared patterns were also observed in BRCTs of TopBP1, suggesting that multimerization may be an important feature of BRCTs. The recombinant second BRCT of XRCC1 (X-ray repair cross-complementing group 1), whose folding was determined by X-ray crystallography, also showed similar DNA end binding images. It is possible that some BRCTs are fundamental structures that detect DNA damages.

Release of allergenic proteins from rice grains induced by high hydrostatic pressure.

Protein release from rice grains during high-pressure treatment was investigated. When polished rice grains were immersed in distilled water and pressurized at 100-400 MPa, a considerable amount of proteins (0.2-0.5 mg per gram of grains) was released. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses, the major proteins released were identified as 16 kDa albumin, alpha-globulin, and 33 kDa globulin, which were known as major rice allergens. By scanning electron microscopic observation of rice grains pressurized at 300 MPa, partial morphological changes in endosperm cells but no apparent structural changes in protein bodies were detected. The content of these allergenic proteins decreased by pressurization and almost completely disappeared from rice grains by the pressurization in the presence of proteolytic enzyme. These results suggest that partial destruction of endosperm cells caused by pressurization enhances permeability of a surrounding solution into rice grains and that a part of the proteins are solubilized and subsequently released into a surrounding solution.

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors.

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Reelin molecules assemble together to form a large protein complex, which is inhibited by the function-blocking CR-50 antibody.

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo. The Reelin-Reelin interaction clearly is inhibited by the function-blocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment.

Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is ubiquitous in several proteins that participate in cell cycle checkpoints and DNA repair. We have previously shown that the BRCT regions of TopBP1 (DNA topoisomerase II binding protein 1) and BRCA1 bound DNA breaks. A BRCT-related region, BRCT-W1, in the retinoblastoma susceptibility gene product (Rb) also could bind DNA fragments, independently of DNA sequences. Five BRCT-W regions were found in the Rb family. All BRCT-Ws of Rb bound DNA fragments. Electron microscopy and treatment with an exonuclease showed that BRCT-Ws bound double-strand DNA breaks. Since some BRCTs are exceptional common relating elements in tumor suppression, our findings reveal novel aspects of the tumor suppression mechanism.

A novel chromosomal locus of enteropathogenic Escherichia coli (EPEC), which encodes a bfpT-regulated chaperone-like protein, TrcA, involved in microcolony formation by EPEC.

The bfpTVW operon, also known as the per operon, of enteropathogenic Escherichia coli (EPEC) is required for the transcriptional activation of the bfp operon, which encodes the major subunit and assembly machinery of bundle-forming pili (BFP). An immobilized T7-tagged BfpT fusion protein that binds specifically to upstream promoter sequences of bfpA and eae was used to 'fish out' from a promoter library other EPEC chromosomal fragments that are bound by the BfpT protein. After screening for promoters exhibiting bfpTVW-dependent expression, one was identified that was positively regulated by bfpTVW and that is not present in the chromosomes of two non-virulent E. coli laboratory strains, DH5alpha and HB101. Further analysis of this positively regulated promoter in EPEC showed that it resided within a 4.9 kb sequence that is not present in E. coli K12. This locus, located downstream of the potB gene, was found to contain four open reading frames (ORFs): bfpTVW-activated promoter was localized upstream of ORF1. An ORF1 knockout mutant produced less of the BFP structural subunit (BfpA) and formed smaller than normal adherent microcolonies on cultured epithelial cells; however, this mutation did not affect bfp transcription. An ORF1-His6 fusion protein specifically bound the preprocessed and mature forms of the BfpA protein and thus appears to stabilize the former within the cytoplasmic compartment. ORF1 therefore is a newly isolated EPEC chromosomal gene that encodes a chaperone-like protein involved in the production of BFP. Hence, ORF1 was designated trcA (bfpT-regulated chaperone-like protein gene). The TrcA protein also specifically bound 39 kDa and 90 kDa proteins that are expressed by EPEC but not by E. coli K12. The 90 kDa protein was revealed to be intimin, a protein product of the eae gene, which is required for the EPEC attaching/effacing phenotype, suggesting a direct interaction of TrcA with intimin in the cytoplasmic compartment.

Inner-arm dynein c of Chlamydomonas flagella is a single-headed processive motor.

Axonemal dyneins are force-generating ATPases that produce movement of eukaryotic cilia and flagella. Several studies indicate that inner-arm dyneins mainly produce bending moments in flagella and that these motors have inherent oscillations in force and motility. Processive motors such as kinesins have high duty ratios of attached to total ATPase cycle (attached plus detached) times compared to sliding motors such as myosin. Here we provide evidence that subspecies-c, a single-headed axonemal inner-arm dynein, is processive but has a low duty ratio. Ultrastructurally it is similar to other dyneins, with a single globular head, long stem and a slender stalk that attaches to microtubules. In vitro studies of microtubules sliding over surfaces coated with subspecies-c at low densities (measured by single-molecule fluorescence) show that a single molecule is sufficient to move a microtubule more than 1 microm at 0.7 microm s(-1). When many motors interact the velocity is 5.1 microm s(-1), fitting a duty ratio of 0.14. Using optical trap nanometry, we show that beads carrying a single subspecies-c motor move processively along the microtubules in 8-nm steps but slip backwards under high loads. These results indicate that dynein subspecies-c functions in a very different way from conventional motor proteins, and has properties that could produce self-oscillation in vivo.

Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus.

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (<5 mM). At higher sucrose concentrations (>10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.

Further characterization of the type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm.

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.

Cooperativity between two heads of dictyostelium myosin II in in vitro motility and ATP hydrolysis.

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 microM actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax. These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity.

Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex.

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.

A hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity.

The motor function of smooth muscle myosin is activated by phosphorylation of the regulatory light chain (RLC) at Ser19. However, the molecular mechanism by which the phosphorylation activates the motor function is not yet understood. In the present study, we focused our attention on the role of the central helix of RLC for regulation. The flexible region at the middle of the central helix (Gly95-Pro98) was substituted or deleted to various extents, and the effects of the deletion or substitution on the regulation of the motor activity of myosin were examined. Deletion of Gly95-Asp97, Gly95-Thr96, or Thr96-Asp97 decreased the actin-translocating activity of myosin a little, but the phosphorylation-dependent regulation of the motor activity was not disrupted. In contrast, the deletion of Gly95-Pro98 of RLC completely abolished the actin translocating activity of phosphorylated myosin. However, the unregulated myosin long subfragment 1 containing this RLC mutant showed motor activity the same as that containing the wild type RLC. Since long subfragment 1 motor activity is unregulated by phosphorylation, i.e. constitutively active, these results suggest that the deletion of these residues at the central helix of RLC disrupts the phosphorylation-mediated activation mechanism but not the motor function of myosin itself. On the other hand, the elimination of Pro98 or substitution of Gly95-Pro98 by Ala resulted in the activation of actin translocating activity of dephosphorylated myosin, whereas it did not affect the motor activity of phosphorylated myosin. Together, these results clearly indicate the importance of the hinge at the central helix of RLC on the phosphorylation-mediated regulation of smooth muscle myosin.

Dynein arms are oscillating force generators.

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists of a regular array of microtubules. Using optical trapping nanometry, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP8, they generate a peak force of approximately 6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of approximately 2 pN and approximately 30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is approximately 70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.

Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell.

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of >5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 degrees C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 degrees C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4-5 nm on target cells.

Quick-freeze deep-etch electron microscopy of the actin-heavy meromyosin complex during the in vitro motility assay.

Since mica is a substitute for glass in the in vitro actin motility assay, I examined the structure of heavy meromyosin (HMM) crossbridges supporting actin filaments by quick-freeze deep-etch replica electron microscopy. This method was capable of resolving the inter-domain cleft of the monomeric actin molecule. HMM heads that are not bound to actin, when observed by this technique, were straight and elongated in the absence of ATP but strongly kinked upon addition of ATP or ADP.inorganic vanadate to produce the putative long-lived analog of HMM-ADP.inorganic phosphate. The low-magnification image of the ATP-containing acto-HMM preparation showed features characteristic of sliding actin filaments on glass coverslips. At high magnification, all the HMM molecules were found attached to actin by one head with the majority projecting perpendicular to the filament axis, whereas in the absence of ATP, HMM exhibited two-head binding with a preponderance of molecules tilted at 45 degrees. Detailed examination of the shape of HMM heads involved in sliding showed a rounded, and flat appearance of the tip and comparatively thin neck portion as if the heads grasp actin filament, in contrast to rigor crossbridges which have a pear-shaped configuration with more gradual taper. Such configurations of HMM heads were essentially the same as I observed previously on acto-myosin subfragment-1 (S1) by the same technique, except for the presence of an additional neck portion of HMM which makes interpretaion of the images easier. Interestingly, under actively sliding conditions, very few heads were tilted in the rigor configuration. At first glance, the addition of ADP to the rigor-complex gave images rather like those obtained with ATP, but they turned out to be different. The contribution of the structural change of crossbridges to the force development is discussed.

Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron microscopy.

Quick-freeze deep-etch replica electron microscopy combined with mica-flake technique provides high contrast, high time- and spatial-resolution images of protein molecules in solution, whose three-dimensional structure is well preserved. Thus, it might be quite useful to obtain structural information of individual functioning molecules, such as myosin crossbridges under in vitro motility assay conditions. With that method, we could actually show that both heads of heavy meromyosin (HMM) crossbridges are mostly straight and bound to actin filaments with about 45 degree tilt-angle under rigor conditions, whereas they attached to actin through only one head with a wide variety of angles under in vitro sliding conditions. We also demonstrated that free HMM heads are strongly kinked in the presence of ATP or ADP/inorganic vanadate (Vi) in contrast to almost straight configuration in the absence of nucleotide. To examine more detailed structure of individual crossbridges, we tried to reconstruct the three-dimensional architecture of intramolecular subdomains of single HMM molecule. We took a series of tilted images of single HMM-ADP/Vi particle and successfully obtained its 3-D image by filtered back-projection, even with restricted range of tilt-angles. By comparison of the reconstruction with the atomic model of subfragment-1 (S1) without nucleotide, we found some great structural difference, which partly might be attributable to the conformational change by nucleotide binding.

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.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.

[Electron microscopy for the studies of membrane transport proteins].

Electron microscopy gives the real images of individual protein molecules, providing unique means in the structural research of membrane-transport proteins. Various techniques for the preparation of specimens for transmission electron microscopy were briefly described. Emphases were put on the recent progress of quick-freeze cryo-electron microscopy, including cryotransfer and freeze-fracture deep-etch replication. Exploiting the advantage of the latter technique, we visualized the in situ structure of inositol 1,4,5-trisphosphate receptor molecules in the smooth-surfaced endoplasmic reticulum (sER) membrane of the Purkinje neuron in bovine cerebellum. The tetrameric receptor molecules were unexpectedly small and formed a two-dimensional crystalline array on the surface of the cisternal stacks derived from sER. Its structural characteristics were discussed in comparison with the ryanodine-receptor, the other intracellular Ca2+ -channel.