Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons.

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes.

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.

Analysis and separation of synaptosomal membrane proteins.

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with two-dimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondrial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized. Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.

Presence of a protein immunologically related to lamin B in the postsynaptic membrane of Torpedo marmorata electrocyte.

The Torpedo electrocyte is a flattened syncytium derived from skeletal muscle, characterized by two functionally distinct plasma membrane domains. The electrocyte is filled up with a transversal network of intermediate filaments (IF) of desmin which contact in an end-on fashion both sides of the cell. In this work, we show that polyclonal antibodies specific for lamin B recognizes a component of the plasma membrane of Torpedo electrocyte. This protein which thus shares epitopes with lamin B has a relative molecular mass of 54 kD, an acidic IP of 5.4. It is localized exclusively on the cytoplasmic side of the innervated membrane of the electrocyte at sites of IF-membrane contacts. Since our previous work showed that the noninnervated membrane contains ankyrin (Kordeli, E., J. Cartaud, H. O. Nghiêm, L. A. Pradel, C. Dubreuil, D. Paulin, and J.-P. Changeux. 1986. J. Cell Biol. 102:748-761), the present results suggest that desmin filaments may be anchored via the 54-kD protein to the innervated membrane and via ankyrin to the noninnervated membrane. These findings would represent an extension of the model proposed by Georgatos and Blobel (Georgatos, S. D., and G. Blobel. 1987a. J. Cell Biol. 105:105-115) in which type III intermediate size filaments are vectorially inserted to plasma and nuclear membranes by ankyrin and lamin B, respectively.

A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

Distribution of Na+ channels and ankyrin in neuromuscular junctions is complementary to that of acetylcholine receptors and the 43 kd protein.

We have used immunogold electron microscopy to study the organization of the acetylcholine receptor, 43 kd protein, voltage-sensitive Na+ channel, and ankyrin in the postsynaptic membrane of the rat neuromuscular junction. The acetylcholine receptor and the 43 kd protein are concentrated at the crests of the postsynaptic folds, coextensive with the subsynaptic density. In contrast, Na+ channels and ankyrin are concentrated in the membranes of the troughs and in perijunctional membranes, both characterized by discontinuous submembrane electron-dense plaques. This configuration of interspersed postsynaptic membrane domains enriched in either Na+ channels or acetylcholine receptors may facilitate the initiation of the muscle action potential. Furthermore, the results support the involvement of ankyrin in immobilizing Na+ channels in specific membrane domains, analogous to the proposed involvement of the 43 kd protein in acetylcholine receptor immobilization.

Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle.

Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin-related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin-related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.

Isolation and reconstitution of the high-affinity choline carrier.

Monoclonal antibodies, which block the high-affinity uptake of choline in synaptosomal ghosts, have been used to purify a membrane polypeptide (80 kDa) from insect synaptosomal membranes. This isolated protein was found to catalyse the sodium-dependent, hemicholinium-sensitive accumulation of choline after reconstitution into liposomes, thus, apparently represents the high-affinity choline transporter.

Co-ordinate control of CDP-choline and phosphatidylethanolamine methyltransferase pathways for phosphatidylcholine biosynthesis occurs in response to change in diet fat.

Brain microsomal and synaptic plasma membrane phosphatidylcholine composition and biosynthetic activity were examined in relation to the composition of diet fat fed. Phosphocholinetransferase and methyltransferase activities are shown to be modulated by the diet, and by changes in the membrane phospholipid content of long-chain polyunsaturated fatty acids. This physiological modulation is co-ordinated such that the rate of phosphatidylcholine synthesis via one route is inversely regulated with activity of the alternate pathway.

[The characteristics of [3H]muscimol binding to the synaptic membranes of the cerebral cortex in the progeny of rats subjected to deprivation of the paradoxical sleep phase during pregnancy]. / Osobennosti sviazyvaniia [3H]mustsimola s sinapticheskimi membranami kory bol'shikh polusharii mozga u potomstva krys, podvergavshikhsia deprivatsii paradoksal'noi fazy sna vo beremennosti.

White rats were subjected to REM-sleep deprivation during 14-20 days of pregnancy. [3H]muscimol binding level of neocortex synaptosomal membranes was significantly higher in their offsprings as compared with the control ones. Possible ways of GABA-ergic system malformations are discussed.

Solubilization and purification of opioid receptor molecules of rat brain.

A P2 membrane preparation of rat brain (without cerebellum) was solubilized with CHAPS in Tris containing DTT and trypsin inhibitor. Two opiate ligands, 10b and 10cd, prepared by Liu et al, were employed consecutively in affinity chromatography, from which OPR's were eluted with Nx. The eluate was subsequently passed through a WGA affinity column and the OPR's eluted with N-GluNAc. This eluate was further purified and concentrated by preparative granular gel isoelectric focusing on SG200. Two protein peaks appeared separately at pH 5 and 7.8. The eluates from both peaks were examined for protein contents using the silver staining method, and binding activity was measured by RRA with 3H-etor. The results revealed that both samples contained active OPR purified to over 80,000 fold. The Mr was estimated by gel filtration to be 52 kD and 42 kD for OPR in the pH 5 and pH 7.8 samples respectively. OPR in the pH 5 sample have been determined to be of mu-type by their binding activity with 3H-ohm.

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1.

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

[Lyophilized membranes as a test system for analyzing the properties of neuromediator receptors and activity of neurotropic agents]. / Liofilizovannye membrany kak test-sistema dlia analiza svoistv retseptorov neiromediatorov i aktivnosti neirotropnykh sredstv.

The effect of lyophilization and prolonged storage of rat brain membrane preparations on the properties of receptors of cholinergic and adrenergic neuromediator systems has been studied by the radioreceptor assay. Lyophilized membrane preparations have been shown to be highly stable as compared with fresh membranes and retain their binding properties during storage for 1 year.

Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes.

Two conformational states of the nicotinic acetylcholine receptor have been investigated by cryoelectron microscopy of flattened vesicular crystals grown from Torpedo marmorata postsynaptic membranes. One was obtained from the vesicles without acetylcholine present, and is presumed to correspond to the native, or resting state; the other was obtained from the vesicles after exposure to 100 microM to 5 mM carbamylcholine (an acetylcholine analogue) and is presumed to correspond to a desensitized state. Both conformations were determined in three-dimensions to a resolution of 18 A, sufficient to reveal the configurations of the five subunits around the central ion channel over most of their length. The subunits of either structure have a similar appearance, consistent with their amino acid homology. They are each aligned almost parallel to the axis of the receptor, conferring a high degree of pentagonal symmetry to the bilayer portion and a contiguous region on the synaptic side. Their external surfaces form a pronounced ridge in the bilayer portion, which broadens toward the synaptic end. Comparison of features in the two three-dimensional maps reveals that carbamylcholine induces a quaternary rearrangement, involving predominantly the delta-subunit. The densities corresponding to this subunit are tilted by approximately 10 degrees tangential to the axis of the receptor over a large fraction of its length, and become misaligned relative to the densities corresponding to the other four subunits. The gamma-subunit is also affected, being displaced slightly away from the axis of the receptor. The alpha- and beta-subunits may be affected on a more localized scale. The overall changes are most pronounced in the synaptic region, where the ligand-binding site is located, and in the cytoplasmic region, which may be closer to the gate of the channel. The physiological process of desensitization appears to be associated with a structural transition in which the subunits switch to a less symmetrical configuration.

Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody.

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent Mr 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the Mr of both glycoproteins by some 5-7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.