Nerve Terminal Protein Research Articles

Distribution of αlatrotoxin receptor in the rat brain by quantitative autoradiography: Comparison with the nerve terminal protein, synapsin I

Tissue slice autoradiography was employed to reveal the brain distribution of the receptor for αlatrotoxin, the presynaptic neurotoxin of the black widow spider venom. The receptor distribution pattern was compared with that of a marker protein for nerve endings, synapsin I, a phosphoprotein known to be present within nerve terminals. The αlatrotoxin receptor and synapsin I were detected in gray matter-containing regions but their relative amounts were not constant. In the cerebral cortex and in the caudatum their distribution was similar, while in the hippocampus they were both abundant, but their distribution varied: synapsin I labeling was heavier in CA4 and CA3,αlatrotoxin receptor labeling in CA1 and dentate gyrus. A dissociation was also observed in the globus pallidus and in the lateral thalamic nuclear complex, where αlatrotoxin receptor labeling was very weak. The most striking dissociation occurred in the cerebellum, where the molecular layer was strongly labeled for synapsin I, but almost unlabeled for the αlatrotoxin receptor, which was more concentrated in the granular layer. Taken as a whole, the data appear compatible with a widespread localization of the αlatrotoxin receptor at synapses. However, they also suggest that either some nerve terminals are insensitive to αlatrotoxin, or the receptor for the toxin is not present at a similar concentration in all presynaptic plasma membranes.

Dopamine-regulated phosphorylation of synaptic vesicle-associated proteins in rat neostriatum and substantia nigra

Dopamine, acting through dopamine D 1 receptors and cyclic AMP-dependent protein kinase, has been found to increase the state of phosphorylation of the synaptic vesicle-associated phosphoproteins synapsin I and protein III in slices of rat neostriatum and substantia nigra. In the neostriatum, the effect of dopamine was mimicked by SKF 38393, a D 1 receptor agonist, and was abolished by preincubation of the slices with fluphenazine or SCH 23390, antipsychotic drugs which are potent D 1 receptor antagonists, but not by the D 2 receptor antagonists l-sulpiride or spiroperidol. The maximal effect of dopamine in the neostriatum represented approximately 30–35% of the maximal effect induced by 8-bromo cyclic AMP, suggesting that a similar fraction of nerve terminals in the neostriatum may express the dopamine D 1 receptor. Evidence for a small population of beta-adrenergic receptors regulating nerve terminal protein phosphorylation in the neostriatum, distinct from the D 1 dopamine receptors, was also obtained. In the substantia nigra, the effect of dopamine also appeared to be mediated through a D 1 dopamine receptor, since it was abolished by fluphenazine and SCH 23390. The maximal effect of dopamine in the substantia nigra represented approximately two-thirds of the effect induced by 8-bromo cyclic AMP, suggesting that a similar fraction of nerve terminals in the substantia nigra may express the dopamine D 1 receptor. The ability of dopamine D 1 receptor activation to stimulate both synapsin I and protein III phosphorylation and GABA release in both the neostriatum and substantia nigra may be causally linked.

Immunological detection of mediatophore in motor end-plates and electric organ subcellular fractions of torpedo marmorata

A rabbit antiserum to mediatophore, a nerve terminal membrane protein involved in calcium dependent ACh release, was raised after immunization with the purified protein. An immunological assay for mediatophore was then developed and the subcellular distribution of this protein in Torpedo electric organ fractions was studied. A good agreement was obtained between the distribution in the different fractions of the antigen and of mediatophore related acetylcholine releasing activity as determined by reconstitution in proteoliposomes. Mediatophore was highly concentrated in presynaptic plasma membranes of electric organ, while very low contents were observed in electric nerves and electric lobes. Although some mediatophore was found in synaptic vesicle fractions, this most probably resulted from presynaptic membrane contamination as evaluated with other presynaptic membrane markers. Nerve terminals of motor end-plates were strongly stained with anti-mediatophore antibodies.

Nerve terminal anchorage protein 1 (TAP-1) is a chondroitin sulfate proteoglycan: biochemical and electron microscopic characterization.

The plasma membranes of the nerve terminal and the postsynaptic cell of electric organ are separated by a basal lamina. We have purified, biochemically characterized, and visualized in the electron microscope a macromolecule which appears to anchor the nerve terminal to this basal lamina. This molecule, terminal anchorage protein 1 (TAP-1) is associated with the nerve terminal membrane of electric organ, has the properties of an integral membrane protein, and is tightly bound to the extracellular matrix (Carlson, S.S., P. Caroni, and R.B. Kelly. 1986. J. Cell Biol. 103:509-520). TAP-1 can be solubilized from an electric organ extracellular matrix preparation with guanidine-HCl/3-[(3-cholamidopropyl)-dimethylammnio]-1-propane sulfonate and purified by a combination of permeation chromatography on Sephacryl S-1000, sedimentation velocity, and ion exchange chromatography on DEAE Sephacel. The total purification from electric organ is 91-fold and results in at least 86% purity. Digestion of the molecule with chondroitin ABC or AC lyase produces a large but similar shift in the molecular weight of the molecule on SDS-PAGE. The presence of chondroitin-4- or 6-sulfate is confirmed by identification of the isolated glycosaminoglycans with cellulose acetate electrophoresis. Gel filtration of the isolated chains indicates an average molecular weight of 42,000. Digestion of TAP-1 with other glycosaminoglycan lyases such as heparitinase indicates that only chondroitin sulfate is present. These results demonstrate that TAP-1 is a proteoglycan. Visualization of TAP-1 in the electron microscope reveals a "bottlebrush" structure expected for a proteoglycan. The molecule has an average total length of 345 +/- 17 nm with 20 +/- 2 side projections of 113 +/- 5 nm in length. These side projections are presumably the glycosaminoglycan side chains. From this structure, we predict that the TAP-1 glycosaminoglycan side chains should have a molecular weight of approximately 50,000, which is in close agreement with the biochemical studies. Both biochemical and morphologic data indicate that TAP-1 has a relative molecular weight of approximately 1.2 X 10(6). The large size of TAP-1 suggests that this molecule could span the synaptic cleft and make a significant contribution to the structure of the nerve terminal basal lamina of electric organ.

A nerve terminal anchorage protein from electric organ.

The nerve terminal and the postsynaptic receptor-containing membranes of the electric organ are both linked to the basal lamina that runs between them. We have identified an extracellular matrix protein whose physical properties suggest it anchors the nerve terminal to the basal lamina. The protein was identified because it shares an epitope with a proteoglycan component of electric organ synaptic vesicles. It too behaves like a proteoglycan. It is solubilized with difficulty from extracellular matrix fractions, elutes from DEAE Sephacel at pH 4.9 only at high ionic strength, and binds to a laminin affinity column from which it can be eluted with heparin. Under denaturing conditions it sediments rapidly and has a large excluded volume although it can be included in Sephacryl S-1000 columns. This large, highly charged extracellular matrix molecule can be readily reconstituted into liposomes consistent with the presence of a hydrophobic tail. By immunoelectron microscopy the antigen is found both in synaptic vesicles and on the plasma membrane of the nerve terminal. Since this is the first protein described that links the nerve terminal membrane to the extracellular matrix, we propose calling it terminal anchorage protein one (TAP-1).

In situ degradation of rapidly transported proteins in nerve terminals of retinal ganglion cells

The in situ degradation of rapidly transported proteins in nerve terminals of retinal ganglion cells was studied in pieces of the superior colliculus of the rabbit. Proteolytic degradation was found to be dependent upon extracellular calcium and intact calcium channels. Degradation was inhibited by leupeptin and SH-group blocking agents.

Calcium-dependent calmodulin binding to cholinergic synaptic vesicles.

Calmodulin is a major nerve terminal protein and a potential mediator of calcium-dependent nerve terminal functions. Calcium-dependent calmodulin binding has been reported in secretory membrane preparations including chromaffin granules and crude rat brain vesicles. Here we demonstrate a calcium-dependent calmodulin-binding site on cholinergic synaptic vesicles from electric organ. It is saturable with high affinity (KD = 10 nM; Bmax = 80 pmol/mg). The binding is inhibited by trifluoperazine (I50 = 8 microM) and is at least 1000-fold specific for calmodulin over troponin C. Association and dissociation rates (k = 3.1 X 10(6) M-1S-1; k-1 = 1.3 X 10(-2) S-1) are consistent with the dissociation constant measured at equilibrium. Intact synaptic vesicles bind to calmodulin immobilized on polyacrylamide matrix, suggesting that the binding site is cytoplasmically oriented in the vesicle population. Intact synaptic vesicles bind calmodulin up to 80-fold more effectively than do side fractions from the vesicle purification. The quantitative difference is largely due to latency of binding sites since it disappears when the binding is assayed in detergent. Binding of calmodulin to proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that a subset of nerve terminal and electric organ calmodulin-binding proteins are found in synaptic vesicles.

ADP-ribosylation of membrane proteins in cholinergic nerve terminals.

Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [32P]NAD+ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of Mr = 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcellular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with Mr = 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nucleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes.

Intracellular transport in neurons.

Intracellular transport in neurons.

Properties of individual nerve terminal proteins identified by two-dimensional gel electrophoresis

Properties of individual nerve terminal proteins identified by two-dimensional gel electrophoresis

Nerve terminal proteins of the rabbit visual relay nuclei identified by axonal transport and two-dimensional gel electrophoresis

The proteins in nerve terminals can be uniquely identified by two-dimensional gel electrophoresis of proteins labeled during synthesis in the cell body and then transported intra-axonally to the terminals. We have explored the potential of the identification procedure by comparing the proteins which are transported from the retina to the lateral geniculate nucleus (LGN) and the superior colliculus (SC) of the rabbit. We have been able to identify between 150 and 200 proteins which ate common to both LGN and SC nerve terminals, very few of which are present at significantly different concentrations in one nucleus relative to the other. The similarity between proteins sent from the retina along two neural pathways subserving different functions illustrates the subtlety of biochemical changes that must underlie physiological differences. Only a small fraction of the labeled proteins are major proteins of the relay nuclei as judged by Coomassie-staining, and some of these arise from in situ nonspecific labeling with blood-borne radioactivity, rather than by transport to the terminals. We have shown that about 5 times more proteins are transported at fast than at intermediate transport rates. More than 50% of the fast proteins turn over rapidly and are gone in 24 h. Few intermediate proteins turn over rapidly. Since only 6% of the proteins in the relay nuclei (at 36 h) could not be detected in the optic tract at that time, transsynaptic labeling by breakdown and resynthesis must be small, if it occurs at all.

Stimulation of Ca 2+ -dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles

Synaptic vesicles have a Ca(2+)-dependent protein kinase system that may play a role in mediating Ca(2+)-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca(2+)-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca(2+) uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca(2+) and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.

CENTRAL EFFECTS OF CENTRIPETAL IMPULSES IN AXONS OF SPINAL VENTRAL ROOTS

CENTRAL EFFECTS OF CENTRIPETAL IMPULSES IN AXONS OF SPINAL VENTRAL ROOTS