Teruo Abe

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.

Effects of synthetic ω-conotoxin on synaptic transmission

Abstract The effects of chemically synthesized ω-conotoxin GVIA (a neurotoxic peptide from Conus geographus ) on synaptic transmission at the bullfrog sympathetic ganglion, frog neuromuscular junction and electric organ of the ray, Narke japonica , were studied. The synthetic toxin irreversibly suppressed synaptic transmission at these synapses by arresting the release of transmission from the nerve terminals without showing postsynaptic effects. This action of the toxin was effectively antagonized by high concentrations of extracellular Ca 2+ . The synthetic toxin irreversibly blocked the Ca 2+ -dependent action potential of bullfrog sympathetic ganglion cells. These results suggest that ω-conotoxin GVIA blocks synaptic transmission by interfering with the Ca 2+ influx through the voltage-sensitive Ca 2+ channel of the nerve terminal. These results indicate that the chemically synthesized ω-conotoxin GVIA acts exactly like the natural ω-conotoxin GVIA. Thus, the synthetic toxin can be used in place of the natural toxin as a useful probe for the voltage-sensitive Ca 2+ channel in the nervous system.

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.

Differential effects of apamin on Ca2+-dependent K+ currents in bullfrog sympathetic ganglion cells

In B-type neurones of bullfrog sympathetic ganglia, apamin (10 nM) suppressed the Ca2+-dependent K+ current (IAH) involved in the afterhyperpolarization of an action potential, while it did not affect the Ca2+-dependent K+ current (Ic) underlying the spike repolarization. IAH was further separated into two exponential components which were differentially affected by apamin, voltage and alterations in Ca2+ influx, suggesting the existence of 3 different types of Ca2+-dependent K+ channel in bullfrog sympathetic neurones.

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.

Cloning and functional expression of a cDNA encoding the mouse β2 subunit of the kainate-selective glutamate receptor channel

The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.

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.

Blockade of [3H]lysine-tetrodotoxin binding to sodium channel proteins by conotoxin GIII.

Conotoxin GIII from Conus geographus inhibited the binding of [3H]lysine-tetrodotoxin (4 nM) to electroplax membranes from Electrophorus electricus and to the rat brain P2 fraction with IC50 values of 13 nM and 7.9 microM, respectively. This inhibition observed with electroplax membranes was irreversible. These and physiological findings (Life Sci., 21 (1977) 1759-1770 suggest that conotoxin GIII inhibits Na channel activation by its interaction with the tetrodotoxin binding site of the Na channel. The differences in structures related to the activation of Na channels between the eel electroplax and the rat brain are indicated.