SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis.

Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.

Immunohistochemical distribution of the two isoforms of synaphin/complexin involved in neurotransmitter release: localization at the distinct central nervous system regions and synaptic types.

The cellular and subcellular localization of the two synaphin isoforms, proteins associated with the docking/fusion complex crucial to neurotransmitter release, was studied in the rat central nervous system by using light microscopic and electron microscopic immunohistochemistry with monoclonal antibodies specific to each isoform. Synaphin 1 (complexin II) was predominantly expressed in neurons of the central nervous system regions such as cerebral cortex (the II, III and VI cortical layers), claustrum, hippocampus, entorhinal cortex, amygdaloid nuclei, substantia nigra pars compacta, superior colliculus, pontine reticulotegmental nucleus and inferior olive, whereas synaphin 2 (complexin I) was in the cerebral cortex (the IV cortical layer), thalamus, locus coeruleus, gigantocellular reticular field, cuneate nucleus and cerebellar basket and stellate cells. In some regions, including the caudate-putamen, globus pallidus, pontine reticular nucleus, cerebellar nuclei and spinal gray matter, synaphin 1 was mainly present in small or medium-sized neurons, while synaphin 2 was in large cells. Medial habenular nucleus and cerebellar granule cells showed both immunoreactivities. In the neuropil of the cerebral cortex and hippocampus, synaphin 1 expression was accentuated in the axon terminals of axospinal and axodendritic synapses, while synaphin 2 was predominant in the axon terminals of axosomatic synapses. In the axon terminals, both immunolabelings were associated with synaptic vesicles and the plasma membrane, being accentuated in the vicinity of synaptic contacts. In the cerebral cortex, both immunoreactivities were also present occasionally in dendrites and dendritic spines, associated with microtubules and the plasma membrane including the postsynaptic densities. These results suggest that the two isoforms of synaphin are involved in synaptic function at the distinct presynaptic regions in the central nervous system, and that some dendrites are another functional site for the proteins.

Distinct regional distribution in the brain of messenger RNAs for the two isoforms of synaphin associated with the docking/fusion complex.

Synaphin is a 19,000 mol. wt cytosolic protein we first found to co-purify with the docking/fusion complex crucial to neurotransmitter release from presynaptic terminals. Two isoforms of synaphin (synaphins 1 and 2) (also called complexins II and I, respectively) exist in the rat brain. On density gradient centrifugation of a Triton X-100 extract of brain membranes, synaphin was found to be associated with the 7S complex that contains synaptotagmin, syntaxin, synaptosomal-associated protein of 25,000 mol. wt and vesicle-associated membrane protein. A smaller complex devoid of synaphins was also identified by immunoprecipitation with a monoclonal antibody against synaptosomal-associated protein of 25,000 mol. wt. Messenger RNAs for synaphins 1 and 2 were expressed predominantly in the brain. In situ hybridization using probes specific to synaphins 1 and 2 indicated that the distribution of their mRNAs was significantly different in brain regions such as olfactory bulb, hippocampus, cerebral cortex, piriform cortex, cerebellum, thalamus and facial nuclei. These results show synaphin as a component of the 7S complex and suggest different physiological implications for the two isoforms.

Molecular cloning of synaphins/complexins, cytosolic proteins involved in transmitter release, in the electric organ of an electric ray (Narke japonica).

Synaphins/complexins are cytosolic proteins associated with the docking/fusion complex crucial to transmitter release. The electric organ of the electric ray Narke japonica contained at least two kinds of synaphins as revealed by immunoblotting. cDNAs for three synaphins were cloned from a cDNA library prepared from the electric lobe where cell bodies of electromotor nerves innervating the electric organ exist. The proteins encoded by these cDNAs were named Nj-synaphins 1a, 1b and 2 on the basis of their high homologies (83-93%) to mammalian synaphins 1 and 2. Nj-Synaphins were immunoprecipitated by an anti-syntaxin monoclonal antibody, together with syntaxin, SNAP-25 and VAMP (synaptobrevin), suggesting the presence of a docking/fusion complex similar to that in the mammalian brain.

A calcium channel from the presynaptic nerve terminal of the Narke japonica electric organ contains a non-N-type alpha 2 delta subunit.

A monoclonal antibody (MCC-1) that recognizes the alpha 2 delta subunit complex of L-type calcium channels from rabbit skeletal muscle membranes partially inhibited the evoked release of acetylcholine from synaptosomes isolated from the electric organ of the marine electric ray, Narke japonica. Digitonin extracts of synaptosomal plasma membranes were subjected to immunoaffinity column chromatography on MCC-1-Sepharose. The purified fraction contained a 170-kDa protein that reacts with MCC-1 and dissociates into smaller polypeptides under reducing conditions. In addition, immunoblotting analysis revealed the existence of syntaxin in the purified fraction, suggesting that the calcium channel forms a complex with syntaxin. However, MCC-1 did not immunoprecipitate an omega-conotoxin GVIA-binding protein. These findings indicate that the 170-kDa protein may be the alpha 2 delta subunit of a calcium channel that is distinct from the omega-conotoxin GVIA-sensitive N-type calcium channel and partially responsible for the calcium influx that triggers the evoked release of acetylcholine.

Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals.

We previously identified a 19 kDa protein associated with the docking/fusion complex involved in neurotransmitter release. A cDNA for this protein was cloned from a bovine brain cDNA library using an oligonucleotide probe based on its partial amino acid sequence. The protein (named synaphin) encoded by the cDNA is a very hydrophilic protein rich in glutamic acid and lysine residues. It lacks any putative transmembrane segments or strongly hydrophobic domains. Immunoblots with antibodies against synaphin detected the protein only in the nervous system among the tissues examined. In brain, it exists mainly in the soluble fraction and is scarce in synaptic vesicles.

Identification of four different forms of syntaxin 3.

cDNAs for four different forms of syntaxin 3 were cloned from a mouse brain cDNA library, and the proteins encoded by these clones were named syntaxin 3A (previous syntaxin 3), 3B, 3C and 3D. Syntaxin 3B contained a different sequence in the carboxyl terminal region from that of syntaxin 3A. The amino terminal region of syntaxin 3C contained an 18 amino acid sequence instead of a 34 amino acid sequence present in syntaxins 3A and 3B. Syntaxin 3D consisted of only 86 amino acids and lacked any putative transmembrane segments. These forms of syntaxin 3 are probably generated by alternative splicing of the primary transcript of syntaxin 3 gene. Cytoplasmic portions of syntaxins 3A and 3B but not of syntaxin 3C or 3D bound to Munc-18/n-sec1.

Impairment of syntaxin by botulinum neurotoxin C1 or antibodies inhibits acetylcholine release but not Ca2+ channel activity.

The involvement of syntaxin, an omega-conotoxin-sensitive Ca2+ channel-associated protein, in acetylcholine release was studied at synapses formed between rat sympathetic neurons in culture. Transmission at these synapses involved omega-conotoxin-sensitive Ca2+ channels because a dose-dependent inhibition was observed when omega-conotoxin was bath-applied. Confocal microscope examination of immunofluorescent staining showed that syntaxin had a similar distribution to synaptic vesicle-associated membrane proteins, synaptophysin and vesicle-associated membrane protein/synaptobrevin-2, indicating that syntaxin molecules are concentrated in the presynaptic terminals. Botulinum neurotoxin C1 applied extracellularly or intracellularly into presynaptic neurons blocked synaptic transmission. Introduction of a monoclonal antibody, or polyclonal antibodies, to syntaxin into the presynaptic neuron depressed the evoked release of acetylcholine without affecting Ca2+ influx through Ca2+ channels. These results suggest that syntaxin plays an important role in release of neurotransmitter by a nerve impulse and that this mechanism is downstream of Ca2+ influx.

A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2 and syntaxins in brain presynaptic terminals.

Two monoclonal antibodies (SPM-1 and SPM-2) immunoprecipitate brain N-type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM-1- and SPM-2-Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin-2, and rab3A/smg25A and SNAP-25, respectively. Since SPM-1 and SPM-2 recognize only syntaxins and the 28 kDa band (rab3A/sm25A and SNAP-25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release.

Monoclonal antibodies immunoprecipitating omega-conotoxin-sensitive calcium channel molecules recognize two novel proteins localized in the nervous system.

Two monoclonal antibodies raised against brain synaptic membranes immunoprecipitated significant fractions of the brain omega-conotoxin receptor (probable omega-conotoxin-sensitive calcium channels) solubilized with digitonin. These antibodies recognized different proteins of 36 kDa and 28 kDa, respectively. Immunoblot analysis of fractions obtained by sucrose gradient centrifugation suggested that these two proteins were not subunits of the omega-conotoxin receptor but were bound to it. These proteins were found to be conserved at least from an amphibian to mammals, and to be present in the nervous system and adrenal medulla among the tissues examined.

The high affinity receptor for omega-conotoxin represents calcium channels different from those sensitive to dihydropyridines in mammalian brain.

A monoclonal antibody recognizing the alpha 2 delta complex of the dihydropyridine (DHP)-sensitive calcium channel of skeletal muscle immunoprecipitated most of the DHP receptor solubilized from bovine and rabbit brains, and bovine cardiac muscle. However, it did not significantly immunoprecipitate the high affinity omega-conotoxin receptor solubilized from these brains. These results indicate that the DHP receptor and the high affinity omega-conotoxin receptor are different molecules in mammalian brain.

Solubilization of the omega-conotoxin receptor associated with voltage-sensitive calcium channels from bovine brain.

The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes.

Identification of the receptor for omega-conotoxin in brain. Probable components of the calcium channel.

Recently omega-conotoxin GVIA was shown to specifically block neuronal and other calcium channels. In this work, an azidonitrobenzoyl derivative of mono-[125I]iodo-omega-conotoxin GVIA was used to identify the components of its receptor site in synaptic plasma membrane by photoaffinity labeling. Components of Mr approximately equal to 310,000, approximately equal to 230,000, and 34,000 were specifically photolabeled. The characteristics of photolabeling of these three components were consistent with those of the specific binding of omega-conotoxin GVIA to synaptic plasma membrane with respect to the effects of metal ions, conventional calcium antagonists, and an agonist (1,4-dihydropyridines, verapamil, and diltiazem, etc.), omega-conotoxins GVIIA and GVIIB. Furthermore, the distribution of these three components in subcellular fractions from rat brain as estimated by photolabeling was in good agreement with that of the specific binding of omega-conotoxin GVIA to its receptor. These findings indicate that the components of Mr approximately equal to 310,000, approximately equal to 240,000, and 34,000 are the receptor for omega-conotoxin GVIA and suggest that these components are constituents of the voltage-sensitive calcium channel in brain. No specific photolabeling was observed in the plasma membrane of human erythrocytes, probably indicating the absence of the receptor for omega-conotoxin GVIA in the membrane.

Binding of omega-conotoxin to receptor sites associated with the voltage-sensitive calcium channel.

The binding of radioiodinated omega-conotoxin GVIA, a probable Ca channel antagonist, to synaptic plasma membranes of rat brain was examined. Two kinds of specific binding sites were found with apparent dissociation constants of 10 pM and 0.5 nM and maximum binding capacities of 0.5 and 3.4 pmol/mg prot., respectively. The binding of the toxin was not affected by high concentrations of Ca antagonists or an agonist, indicating distinct binding sites of the toxin from those of these drugs. Divalent and trivalent metal ions strongly inhibited the binding. The order of their inhibitory potencies was similar to that for inhibition of the Ca current through certain Ca channels. These results suggest that the binding sites of omega-conotoxin GVIA are functionally related to the Ca2+-binding site postulated to be in the pore of the Ca channel.

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica.

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.

Detergent-soluble form of acetylcholinesterase in the electric organ of electric rays. Its isolation, characterization and monoclonal antibodies.

The detergent-soluble form of acetylcholinesterase was purified from the electric organ of the electric rays Narke japonica and Torpedo californica, and its properties were examined. The electric organ of N. japonica and T. californica contains three types of acetylcholinesterase: low-salt-soluble, asymmetric or tailed, and detergent-soluble forms. Results showed that in N. japonica, asymmetric forms were predominant, whereas in T. californica the detergent-soluble form was predominant. Low-salt-soluble acetylcholinesterase constituted 10% of the total acetylcholinesterase in both species. Detergent-soluble acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. Triton X-100 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted quantitatively by lowering the pH to 2.8. This simple procedure gave good yields. The purified enzymes gave single peaks at 6 S on sucrose gradients in the presence of detergent and polydisperse aggregates in the absence of detergent. Reduction of disulfide bonds gave peaks at 4.4 S. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the purified acetylcholinesterases gave bands with Mr of about 130 000 in the unreduced state and with Mr of 66 000 in addition to a very faint band of Mr 130 000 in the reduced state. The Mr-66 000 polypeptides were labeled with diisopropylfluorophosphate. Thus, the detergent-soluble acetylcholinesterases exist as dimers of the Mr-66 000 components. Two-dimensional electrophoresis of the purified enzymes indicated their homogeneity. The isoelectric points of both enzymes were 5.1 under the conditions employed. The two enzymes had very similar amino acid compositions, and contained more than 14% of neutral sugars and glucosamine. Monoclonal antibodies were raised to detergent-soluble acetylcholinesterase by the hybridoma technique; eight were obtained. All of them recognized the catalytic subunits of detergent-soluble and asymmetric acetylcholinesterase, and reacted only with detergent-soluble acetylcholinesterase in immunoblots. Four of the monoclonal antibodies inhibited the activities of both the detergent-soluble and asymmetric forms of acetylcholinesterase.

A monoclonal antibody against catalytic subunits of acetylcholinesterase in the electric organ of an electric ray, Narke japonica.

A hybridoma cell line secreting a monoclonal antibody against catalytic subunits of acetylcholinesterase in the electric organ of Narke japonica has been obtained by using a mouse immunized with the nerve terminal membranes prepared from the electric organ of Narke japonica. This monoclonal antibody (Nj-501) reacted with asymmetric, low salt-soluble and detergent-soluble forms of acetylcholinesterase of the electric organ of Narke japonica. Immunoblot analysis showed that the catalytic subunits of the asymmetric, low salt-soluble and detergent-soluble forms of acetylcholinesterase were polypeptides of molecular weight (Mr) 70K, 70K / 66K and 66K, respectively. Nj-501 markedly inhibited the activity of all these forms of acetylcholinesterase.