Hideo Saisu

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.

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

Abstract The binding of radioiodinated ω-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 ω-conotoxin GVIA are functionally related to the Ca 2+ -binding site postulated to be in the pore of the Ca channel.

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/smg25A 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.

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.

Monoclonal antibodies immunoprecipitating ω-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.

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.

Direct, Ca2+-dependent Interaction between Tubulin and Synaptotagmin I A POSSIBLE MECHANISM FOR ATTACHING SYNAPTIC VESICLES TO MICROTUBULES

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca2+, and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 μm tubulin. The Ca2+ dependence mainly resulted from Ca2+ binding to the Ca2+ ligands of synaptotagmin I. The C-terminal region of β-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca2+. These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca2+ concentrations.

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.

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.

The high affinity receptor for ω-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.