Immunogold localization of inositol 1, 4, 5-trisphosphate (InsP3) receptor in mouse cerebellar Purkinje cells using three monoclonal antibodies

Ultrastructural localization of InsP3 receptor in mouse cerebellar Purkinje cells was investigated by immunogold technique using three monoclonal antibodies (mab 10A6, 4C11 and 18A10). The epitopes of the three antibodies were numerously detected on the smooth endoplasmic reticulum (ER) (especially, on the stacks of flattened smooth ER, subsurface cisterns and spine apparatus), scantily on the rough ER and on the outer nuclear membrane, but were not detectable on either the plasmalemma, synaptic densities, mitochondria or Golgi apparatus. Not only mab 4C11 and 10A6 which bind to the N-terminal region of the receptor but also 18A10 which binds to the C-terminal region were localized on the cytoplasmic surface of the ER membranes. This indicates that the C terminus of InsP3 receptor is localized on the cytoplasmic surface of the ER. We noticed that gold particles are usually localized on the fuzzy structure of the cytoplasmic surface of smooth ER, which is suggested to correspond to the feet structure of the ryanodine receptor. In the Nissl body, gold particles were found not only on the ER membranes but also in the cytoplasmic matrix between the rough ER cisterns. We suggest that the peculiar structure of Nissl body, which is composed of parallel cisterns of rough ER, sandwiching a number of free polyribosomes between the cisternal elements, is due to the fact that the major proteins like InsP3 receptor are synthesized mostly on the free polyribosomes and become membrane bound only at the later stage of the biosynthesis.     

Rough surfaced smooth endoplasmic reticulum in rat and mouse cerebellar Purkinje cells visualized by quick-freezing techniques

The in vivo structure of the smooth endoplasmic reticulum (ER) was visualized in rat and mouse cerebellar Purkinje cells by using quick-freezing techniques followed by freeze-substitution for ultrathin-sectioning or freeze-fracturing and deep-etching for replicas. High magnification electron microscopy of the ultrathin sections revealed a surprising finding that all the smooth ER are apparently rough surfaced, and heavily studded with a large number of small dense projections. In the soma the smooth ER appears to be similar to its rough counterpart, except that the projections are slightly smaller, less electron dense and less protrusive on the ER membranes than the ribosomes. The projections were short rectangles, 20 x 20 x 6 nm3 in size, covering the cytoplasmic surface of the smooth ER in a checker-board manner where closely packed. After freeze-etching and replication, they appeared to be composed of four subparticles, surrounding a central channel. Thus the projections are very similar to the foot structure (ryanodine receptor) of the sarcoplasmic reticulum. Furthermore, they were distributed exclusively in the ER compartment and were highly concentrated especially in the smooth ER. This localization of the projections coindides with the intracellular distribution of the inositol 1,4,5-trisphosphate (IP3) receptor determined by quantitative immunogold electron microscopy. These findings would suggest that the projections are tetramers of IP3 receptor molecules and could be used as a morphological marker for the smooth ER in Purkinje cells, which spreads from the soma to the axon and dendrite, up to the tips including the spines. In Purkinje cells tubular smooth ER runs freely in a serpentine fashion or are intertwined to make large membraneous tangles without forming cisternal stacks. It is highly probable that the ER cisternal stacks do not exist naturally in Purkinje cells but are formed artificially during the various procedures for chemical fixation.     

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.     

Subsurface cisterns in the Purkinje cells of cerebellum of Syrian hamster

Summary Three types of subsurface cisterns were observed in Purkinje cells of the cerebellum of the Syrian hamster. The type-1 cisterns are subsynaptic, related to axosomatic synapses, and are separated from the postsynaptic cell membranes with distances of 400–800 Å. These are probably modified rough surfaced endoplasmic reticulum. The type-2 cisterns are closely apposed to the surface membranes of Purkinje cells, and have very little intracisternal space except at the dilated lateral edges. The type-3 cisterns are similar in structure to the type-2 cisterns but in addition are closely associated with mitochondria. The type-2 and type-3 cisterns appear between one and two weeks after birth and are still present in adults, having almost the same frequency of occurrence. Thin cell processes opposite the type-2 and type-3 cisterns are considered to be glial cell processes. The morphological details of these types of subsurface cisterns are described here, and their possible functional significance is briefly discussed.This work was carried out at the Department of Anatomy, University of Minnesota, Minneapolis, USA, and was supported by grants from the China Medical Board of New York and Anatomical Training Grant GM114 from the USPHS.Dr. Takahashi wishes to express his sincere thanks to Dr. A. Lazarow and Dr. R. L. Wood of the Department of Anatomy, University of Minnesota, who enabled him to use facilities for electron microscopy.     

The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R

Members of the Bcl-2 protein family modulate outer mitochondrial membrane permeability to control apoptosis. However, these proteins also localize to the endoplasmic reticulum (ER), the functional significance of which is controversial. Here we provide evidence that anti-apoptotic Bcl-2 proteins regulate the inositol 1,4,5-trisphosphate receptor (InsP(3)R) ER Ca(2+) release channel resulting in increased cellular apoptotic resistance and enhanced mitochondrial bioenergetics. Anti-apoptotic Bcl-X(L) interacts with the carboxyl terminus of the InsP(3)R and sensitizes single InsP(3)R channels in ER membranes to low [InsP(3)], enhancing Ca(2+) and InsP(3)-dependent regulation of channel activity in vitro and in vivo, reducing ER Ca(2+) content and stimulating mitochondrial energetics. The pro-apoptotic proteins Bax and tBid antagonize this effect by blocking the biochemical interaction of Bcl-X(L) with the InsP(3)R. These data support a novel model in which Bcl-X(L) is a direct effector of the InsP(3)R, increasing its sensitivity to InsP(3) and enabling ER Ca(2+) release to be more sensitively coupled to extracellular signals. As a consequence, cells are protected against apoptosis by a more sensitive and dynamic coupling of ER to mitochondria through Ca(2+)-dependent signal transduction that enhances cellular bioenergetics and preserves survival.     

Impaired calcium release in cerebellar Purkinje neurons maintained in culture

Cerebellar Purkinje neurons demonstrate a form of synaptic plasticity that, in acutely prepared brain slices, has been shown to require calcium release from the intracellular calcium stores through inositol trisphosphate (InsP(3)) receptors. Similar studies performed in cultured Purkinje cells, however, find little evidence for the involvement of InsP(3) receptors. To address this discrepancy, the properties of InsP(3)- and caffeine-evoked calcium release in cultured Purkinje cells were directly examined. Photorelease of InsP(3) (up to 100 microM) from its photolabile caged analogue produced no change in calcium levels in 70% of cultured Purkinje cells. In the few cells where a calcium increase was detected, the response was very small and slow to peak. In contrast, the same concentration of InsP(3) resulted in large and rapidly rising calcium responses in all acutely dissociated Purkinje cells tested. Similar to InsP(3), caffeine also had little effect on calcium levels in cultured Purkinje cells, yet evoked large calcium transients in all acutely dissociated Purkinje cells tested. The results demonstrate that calcium release from intracellular calcium stores is severely impaired in Purkinje cells when they are maintained in culture. Our findings suggest that cultured Purkinje cells are an unfaithful experimental model for the study of the role of calcium release in the induction of cerebellar long term depression.     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Cell fine structures observed by scanning electron microscopy]

The scanning electron microscope (SEM) provides vivid seemingly three dimensional images which are easier to understand for us than transmission electron microscopic images. For this point of view scanning electron microscopy is advantageous in morphological researches of cell fine structures. Nevertheless, there were few studies in this field, because SEM had much lower resolution than transmission electron microscope (TEM) and because there was no adequate method to reveal intracellular structures. In recent years, however, the resolution of SEM has been markedly improved and the specimen preparation techniques have also advanced. In this paper, some of our preparation technique for revealing cell surface structures or intracellular structures, in particular, osmium-DMSO-osmium method, and the results observed by these methods were described. 1) Nucleus. The nucleus was wrapped with a nuclear envelope that consisted of two membranes enclosing a narrow space. On the surface of the envelope many nuclear pores were observed. 2) Endoplasmic reticulum (ER). Rough ER consisted of flattened cisternae, arranged in parallel. The surface were studded with many ribosomes which were often arranged spirally to form polysomes. Smooth ER consisted of tubules. 3) Golgi complex. a) The Golgi stacks were all linked by anastomosing. b) Connection between Golgi stacks and rough ER was often observed. c) Cisternae in a Golgi stack were connected each other. 4) Mitochondria. The mitochondrion was bounded by 2 sheets of unit membrane and the inner membrane projected into the interior of the organelles to make mitochondrial cristae.     

Expression and distribution of InsP(3) receptor subtypes in proliferating vascular smooth muscle cells

The expression and distribution of types 1, 2, and 3 inositol 1,4, 5-trisphosphate receptor (InsP(3)R) in proliferating, primary cultures of rat aortic smooth muscle were compared to fully developed and differentiated rat aortic smooth muscle. Subtype-specific InsP(3)R antibodies revealed that the expression of type 1 InsP(3)R was similar in cultured aortic cells and aorta homogenate but expression of type 2 and 3 InsP(3)R subtypes was increased 3-fold in cultured aortic cells. The distribution of the type 1 InsP(3)R was located throughout the cytoplasm; type 2 InsP(3)R was found closely associated with the nucleus and at the plasma membrane; type 3 InsP(3)R was distributed predominantly around the nucleus. Alterations in InsP(3)R subtype expression and localization may have important functions in regulating intracellular calcium release around the nucleus when vascular smooth muscle cells switch to a more proliferating phenotype.     

Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-trisphosphate from Chenopodium rubrum

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor located on the endoplasmic reticulum (ER) triggers an efflux of calcium release from the ER. Sites that bind InsP(3) with high affinity and specificity have also been described in plant cells, but their precise intracellular locations have not been conclusively identified. In contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of this work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membranes were fractionated by sucrose density gradient centrifugation in the presence and absence of Mg(2+) and alternatively by free-flow electrophoresis. An ER-enriched fraction was also prepared. The following enzymes were employed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophosphatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. In all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstrate that the previously characterized InsP(3)-binding site from C. rubrum is localized on the ER. This finding supports previous suggestions of an alternative non-vacuolar InsP(3)-sensitive calcium store in plant cells.     

Antibodies to the Golgi complex and the rough endoplasmic reticulum

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation.     

STUDIES ON THE ENDOPLASMIC RETICULUM : V. Its Form and Differentiation in Pigment Epithelial Cells of the Frog Retina

Pigment epithelial cells of the frog's retina have been examined by methods of electron microscopy with special attention focused on the fine structure of the endoplasmic reticulum and the myeloid bodies. These cells, as reported previously, send apical prolongations into the spaces between the rod outer segments, and within these extensions, pigment migrates in response to light stimulation. The cytoplasm of these cells is filled with a compact lattice of membrane-limited tubules, the surfaces of which are smooth or particle-free. In this respect, the endoplasmic reticulum here resembles that encountered in cells which produce lipid-rich secretions. The myeloid bodies comprise paired membranes arranged in stacks shaped like biconvex lenses. At their margins the membranes are continuous with elements of the ER and in consequence of this the myeloid body is referred to as a differentiation of the reticulum. The paired membranes resemble in their thickness and spacings those which make up the outer segments; they are therefore regarded as intracellular photoreceptors of possible significance in the activation of pigment migration and other physiologic functions of these cells. The fuscin granules are enclosed in membranes which are also continuous with those of the ER. The granules seem to move independently of the prolongations in which they are contained. The report also describes the fine structure of the terminal bar apparatus, the fibrous layer intervening between the epithelium and the choroid blood vessels, and comments on the functions of the organelles depicted.     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

SUBSURFACE CISTERNS AND THEIR RELATIONSHIP TO THE NEURONAL PLASMA MEMBRANE

Subsurface cisterns (SSC's) are large, flattened, membrane-limited vesicles which are very closely apposed to the inner aspect of the plasma membranes of nerve cell bodies and the proximal parts of their processes. They occur in a variety of vertebrate and invertebrate neurons of both the peripheral and central nervous systems, but not in the surrounding supporting cells. SSC's are sheet-like in configuration, having a luminal depth which may be less than 100 A and a breadth which may be as much as several microns. They are separated from the plasmalemma by a light zone of ~50 to 80 A which sometimes contains a faint intermediate line. Flattened, agranular cisterns resembling SSC's, but structurally distinct from both typical granular endoplasmic reticulum (ER) and from Golgi membranes, also occur deep in the cytoplasm of neurons. It is suggested that membranes which are closely apposed may interact, resulting in alterations in their respective properties. The patches of neuronal plasmalemma associated with subsurface cisterns may, therefore, have special properties because of this association, resulting in a non-uniform neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology and metabolism is discussed.     

Subsurface cisterns and lamellar bodies: Particular forms of the endoplasmic reticulum in the neurons

Summary Structures identified as subsurface cisterns (SSC's) and lamellar bodies (LB's) have been observed in the neurons, but not in the glial cells, of the rat and cat substantia nigra. The SSC's are most often opposite what appears to be glial cells, but they are also subsynaptic in position. A single, large (0.4–1.5 ), unfenestrated, usually flattened cistern closely underlies the inner aspect of the plasma membrane of the perikaryon and proximal parts of the neuronal processes at a regular interval ranging about 100–130 Å. They are sheet-like or discoid in configuration and consists of a pentalaminar structure which usually widens at its lateral edges where its membranes are continuous with each other or with rough ER profiles. Filaments, about 70 Å thick, bridge the cleft between the SSC and the overlying plasmalemma. One or more ER cisterns devoid of ribosomes except on their outermost membrane may be stacked up parallel to an SSC and immediately subjecent to it. A dense filamentous network intervenes between the SSC and its closely applied ER cisterns. At higher magnification, it is seen to consist of a finely textured material which is apparently composed of loosely packed tiny particles. These constituent subunits in turn may represent transverse sections of very fine filaments rather than granules. A mitochondrion frequently occurs in the immediate vicinity of an SSC and may be closely applied to its deep surface. Stacks of unfenestrated, parallel, regularly spaced (about 300–400 Å) cisterns, designated lamellar bodies, appear deeper in the karyoplasm. They are most often flattened and appear as pentalaminar structures. These cisterns, as well as the dense filamentous network intervening between them, are structurally similar to those closely applied to SSC's. They are also devoid of ribosomes except on their outermost surfaces. Whorls of similar cisterns are also occasionally observed. Another particular feature of the rough ER consists of the close apposition of two cisterns without any ribosome attached to the inner membranes of the latter structure. It evokes a simplified type of LB's. It is of particular interest to point out that all these cisterns, i.e. the SSC's, their closely applied cistern(s) and those forming the LB's, are connected to the RER membranes, so that a continuous channel occurs between the nuclear membrane and the SSC which closely underlies the plasma membrane. Our observations show that the SSC's and the LB's are structurally related forms of the ER. A parallel may be drawn between the SSC and the lateral element(s) of a dyad (triad). The structural complex consisting of an SSC, the overlying plasmalemma and the cross-bridges linking them, indeed, bears some resemblance to a dyad. It is suggested that membranes which are closely applied may interact, resulting in alterations in their respective properties. These patches of the neuronal plasma membrane associated with SSC's may, therefore, have special properties because of this relationship, resulting in a non-uniform spread of an action potential on the neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology, as well as of the latter structures and LB's in relation to cell metabolism, is to be discussed.This work was supported by grant MA-3448 from the Medical Research Council of Canada. The skilful technical assistance of Mrs. Marjolaine Turcotte is gratefully acknowledged.     

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Cytochemical analysis of the reconstitution of endoplasmic reticulum after microinjection of rat liver microsomes into Xenopus oocytes

Fragments of rough and smooth endoplasmic reticulum purified from rat liver were injected into Xenopus oocyte cytoplasm. Light and electron microscopy, cytochemistry, immunocytochemistry, and enzyme assay were employed to determine the fate of heterologous membranes in the host cytoplasm. The in vivo-incubated microsomes disappeared in a time-dependent manner. Within 3 hr, rough microsomes were replaced by flattened ER cisternae and smooth microsomes were replaced by a network of anastomosing tubules. Polyclonal antibodies against rat liver microsomes and protein A-gold complexes were applied to glycol methacrylate sections of microinjected oocytes. Specific labeling was observed over discrete rough and smooth ER cisternae 3 hr after microinjection. Endogenous ER was not labeled by this technique, and label was not observed when sections were treated with pre-immune antibodies. Diaminobenzidene cytochemistry of microinjected rat lacrimal gland microsomes revealed enzyme activity in heterologous microsomes after 3 hr of in vivo incubation. Control injected microsomes (inactivated by heat denaturation) became associated with autophagic vacuoles, coincident with changes in lysosomal activity. Freshly isolated un-denatured microsomes did not provoke changes in lysosomal activity, and glucose-6-phosphatase activity associated with microinjected membranes could be detected 21 hr after in vivo incubation. Since rat liver microsomes reconstitute after in vivo incubation into cytoplasmic structures resembling those from which they were derived, we conclude that the microinjected membrane fragments act as templates for their own three-dimensional organization.     

Functional coupling of chromogranin with the inositol 1,4,5-trisphosphate receptor shapes calcium signaling

Chromogranins A and B are high capacity, low affinity calcium (Ca(2+)) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP(3) R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP(3) R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP(3)R. Responses were monitored at the single channel level and in intact cells. When using InsP(3) R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP(3) and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca(2+) signaling. These results show that the InsP(3)R/chromogranin interaction amplifies Ca(2+) release from the ER and that chromogranin is an essential component of this intracellular channel complex.     

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.