Mammalian neuromuscular transmission blocked by funnel web toxin.
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Animals↗
Biomedical subjects
M Sugimori
Publications and source records attributed to M Sugimori.
Microdomains of high calcium concentration in a presynaptic terminal.
Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion, supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation of a low-affinity calcium-binding site at the active zone.
Aequorin↗
P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.
We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.
Action Potentials↗
Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system.
In addition to the three types of voltage-dependent calcium channels presently recognized in the CNS, the L-, the T- and the N-types, a fourth distinct type known as the P-type channel has recently been described. This channel, initially recognized in Purkinje cells (and thus the name), is not blocked by dihydropyridines or by omega-conotoxin (GVIA), but is blocked by native funnel-web spider venom and by a polyamine (FTX) extracted from such venom. In addition, a synthetic polyamine (sFTX) has been produced that also specifically blocks P-channels in brain slices and at the neuromuscular junction, and blocks presynaptic Ca2+ currents in other vertebrate and invertebrate forms, as well as channels expressed in Xenopus oocytes following CNS mRNA injections. Using sFTX to form an affinity gel, a protein was isolated and reconstituted into lipid bilayers where it manifests single-channel properties that are electrophysiologically and pharmacologically similar to those of the native P-channels. Rabbits immunized with the isolated protein produced a polyclonal antibody that gave a positive western blot with the purified P-channel protein and generated a reaction product at specific sites in the CNS that agree with the physiological distribution of P-channel activity.
Animals↗
P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells.
The pharmacological and single-channel properties of Ca2+ channels were studied in the somata and dendrites of adult cerebellar Purkinje cells. The Ca2+ channels were exclusively of the high threshold type: low threshold Ca2+ channels were not found. These high threshold channels were not blocked by omega-conotoxin GVIA and were inhibited rather than activated by BAY K 8644. They were therefore pharmacologically distinct from high threshold N- and L-type channels. Funnel web spider toxin was an effective blocker. The channels opened to conductance levels of 9, 14, and 19 pS (in 110 mM Ba2+). These slope conductances were in the range of those reported for N- and L-type channels. Our results are in agreement with previous reports suggesting that Ca2+ channels in Purkinje cells can be classified as P-type channels according to their pharmacology. The results also suggest that distinctions among Ca2+ channel types based on the single-channel conductance are not definitive.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Presynaptic calcium concentration microdomains and transmitter release.
n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.
Aequorin↗
FMRFamide-related peptides potentiate transmission at the squid giant synapse.
The stellate ganglion of the squid Loligo pealli contains the neuropeptides Phe-Met-Arg-Phe-NH2 (FMRFamide), Phe-Leu-Arg-Phe-NH2 (FLRFamide) and at least one N-terminally extended FMRFamide-related peptide that is yet to be fully characterized. Both local application and arterial perfusion of FLRFamide potentiate transmission at the giant synapse. The N-terminally related peptide Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFLRFamide) produced a similar effect. The threshold for both the tetra- and the hepta-peptides was less than 10 microM. Potentiation could be detected as an increase in rate of rise of the EPSPs, as an increase in amplitude of the EPSP in the absence of spikes, or under voltage clamp as an increase in the EPSC. The effect was most pronounced when the synapse was fatigued by high frequency stimulation. Another molluscan peptide, eledoisin and also leucine enkephalin were without effect. In the absence of any detectable effects of FLRFamide on the resting membrane potential of either pre- or postsynaptic terminals or on the presynaptic spike, it is suggested that the peptide influences transmitter mobilization. However, the peptide could also exert small changes in preterminal calcium currents, which so far we have been unable to detect.
Animals↗
Antitumor agents. V. Synthesis and antileukemic activity of E-ring-modified (RS)-camptothecin analogues.
Several E-ring-modified analogues of (RS)-camptothecin were synthesized by total synthesis via Friedländer condensation and evaluated for cytotoxicity and antitumor activity against P388 mouse leukemia cells. Among them, (RS)-20-deoxyamino-7-ethyl-10-methoxycamptothecin (25c) was found to be more active than (RS)-camptothecin (1) in the in vivo assay.
Animals↗
[Tuberculous meningitis in Japanese children between 1980-1991].
We evaluated the clinical and laboratory findings of tuberculous meningitis admitted to Tokyo Metropolitan Children's Hospital from 1980 through 1991 retrospectively. They consisted of 26 patients (14 boys and 12 girls), and their age ranged from 4 months to 11 years. Seventeen patients were under 3 years of age. Mortality rate was 4% (1/26), and there were 13 patients with sequelae on discharge from the hospital, and 12 patients without any sequelae. The period to recover normal CSF findings was relatively long. Some cases which did not seem to respond well to chemotherapy at the initial stage, recovered from meningitis without sequelae. This suggests that it is not necessary to change the chemotherapy at the initial few weeks of therapy. The risk factors of poor prognosis were; age less than 2 years, decreased level of consciousness on admission, convulsion, CSF protein more than 70 mg/dl, and CSF glucose less than 20 mg/dl. Tryptophan reaction was not always positive. Chloride in CSF was not so important to diagnose tuberculous meningitis in children. For the proper diagnosis of tuberculous meningitis in children one should consider several factors such as tuberculin skin test, family history, chest X-ray findings and CSF study. BCG was inoculated in six children (three patients were under one year old and the rests were older than 4 years). In three patients under one year old BCG was seemed to be inoculated after Mycobacterium tuberculosis infection, and apparently not effective to prevent tuberculosis meningitis. We conclude that BCG vaccination is necessary in early infancy to prevent tuberculous meningitis.
Child↗
[Active tuberculosis in children who received INH chemoprophylaxis].
Twelve children who developed active tuberculosis even after receiving isoniazid (INH) chemoprophylaxis were seen at Tokyo Metropolitan Children's Hospital from 1982 through 1991. All cases received INH more than 9 mg/kg/day, except for one case in which the amount of INH administered at the referring hospital was unknown and Streptomycin was administered together with INH. The age of starting INH prophylaxis ranged from 2 months to 13 years, and the age at which clinical symptoms and/or laboratory evidences of active tuberculosis were first manifested ranged from 4 months to 18 years. Five patients developed active tuberculosis after the completion of chemoprophylaxis and patients during chemoprophylaxis, with the first presentation ranging from primary complex (seven), chronic pulmonary tuberculosis (two), tuberculous meningitis (two), and tuberculous pleuritis (one). None of the Mycobacterium tuberculosis resistant to INH was isolated. Reviewing these patients, eleven cases had at least one of the following factors: (1) age less than two years old (2) infectious sources expectorated more Mycobacterium tuberculosis (3) delay in starting INH. Above factors should be considered in initiating INH chemoprophylaxis and subsequent follow-up of the patients.
Adolescent↗
Localization of P-type calcium channels in the central nervous system.
The distribution of the P-type calcium channel in the mammalian central nervous system has been demonstrated immunohistochemically by using a polyclonal specific antibody. This antibody was generated after P-channel isolation via a fraction from funnel-web spider toxin (FTX) that blocks the voltage-gated P channels in cerebellar Purkinje cells. In the cerebellar cortex, immunolabeling to the antibody appeared throughout the molecular layer, while all the other regions were negative. Intensely labeled patches of reactivity were seen on Purkinje cell dendrites, especially at bifurcations; much weaker reactivity was present in the soma and stem segment. Electron microscopic localization revealed labeled patches of plasma membrane on the soma, main dendrites, spiny branchlets, and spines; portions of the smooth endoplasmic reticulum were also labeled. Strong labeling was present in the periglomerular cells of the olfactory bulb and scattered neurons in the deep layer of the entorhinal and pyriform cortices. Neurons in the brainstem, habenula, nucleus of the trapezoid body and inferior olive and along the floor of the fourth ventricle were also labeled intensely. Medium-intensity reactions were observed in layer II pyramidal cells of the frontal cortex, the CA1 cells of the hippocampus, the lateral nucleus of the substantia nigra, lateral reticular nucleus, and spinal fifth nucleus. Light labeling was seen in the neocortex, striatum, and in some brainstem neurons.
Animals↗
Properties of calcium channels isolated with spider toxin, FTX.
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Animals↗
Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse.
1. Presynaptic or simultaneous pre- and postsynaptic voltage-clamp protocols were implemented in the squid giant synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.
Action Potentials↗
Real-time imaging of calcium influx in mammalian cerebellar Purkinje cells in vitro.
Real-time visualization of intracellular calcium concentration ([Ca2+]i) changes in mammalian Purkinje cells in vitro, utilizing the dye Fura-2, indicates that calcium action potentials are generated in the dendritic tree and follow a particular activation sequence. During spontaneous oscillations or after direct current injection, dendritic spikes are initiated as slow and graded plateau potentials at the level of the tertiary or spiny branchlets of the dendrite. As the plateau potentials become sufficiently high to reach the firing threshold for full dendritic spike generation, calcium entry is observed at the more proximal branches of the dendritic tree. These action potentials are then conducted orthodromically toward the soma and may invade other branches in the arbor antidromically. Simultaneous recording of the intracellular electrical activity and the Fura-2 fluorescent signal indicates that the intracellular calcium transients are accompanied by a very rapid increase in intracellular calcium concentration. This increase in [Ca2+]i exhibits an almost equally fast return to baseline after the termination of the action potential, indicating the presence of very efficient calcium sequestering and extruding mechanisms in the dendrites. Iontophoretic application of glutamate at the dendritic level provided a further demonstration of the spatial separation of plateau potentials from dendritic spikes and gives further insights into the details of dendritic integration in this neuron.
Action Potentials↗
Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse.
The molecular events that control synaptic vesicle availability in chemical synaptic junctions have not been fully clarified. Among the protein molecules specifically located in presynaptic terminals, synapsin I and calcium/calmodulin-dependent protein kinase II (CaM kinase II) have been shown to modulate evoked transmitter release in the squid giant synapse. In the present study, analysis of synaptic noise in this chemical junction was used to determine whether these proteins also play a role in the control of spontaneous and enhanced spontaneous transmitter release. Injections of dephosphorylated synapsin I into the presynaptic terminal reduced the rate of spontaneous and enhanced quantal release, whereas injection of phosphorylated synapsin I did not modify such release. By contrast CaM kinase II injection increased enhanced miniature release without affecting spontaneous miniature frequency. These results support the view that dephosphorylated synapsin I "cages" synaptic vesicles while CaM kinase II, by phosphorylating synapsin I, "decages" these organelles and increases their availability for release without affecting the release mechanism itself.
Animals↗
ATP-dependent directional movement of rat synaptic vesicles injected into the presynaptic terminal of squid giant synapse.
The question as to whether synaptic vesicles prepared from vertebrate brain can be transported to the active zones of the squid giant synapse was studied by using a combined optical and electrophysiological approach. In order to visualize the behavior of the vertebrate synaptic vesicles in situ, synaptic vesicles isolated from rat brain were labeled with a fluorescent dye (Texas red) and injected into the presynaptic terminal of the squid giant synapse. The pattern of fluorescence that would result from passive diffusion was determined by coinjection of an unconjugated fluorescent dye (fluorescein). The patterns obtained with fluorescent synaptic vesicles were strikingly different from that obtained by simple diffusion of fluorescein. Although the fluorescein diffused freely in both directions, the vesicles moved preferentially into the terminal--i.e., toward the release sites--at a rate of 0.5 microns/sec. The final distribution of the injected fluorescent synaptic vesicles displayed a discrete localization that suggested a distribution coincident with the active zones of the presynaptic terminal. Like fast axonal transport, but unlike fluorescein movements in the terminal, the vesicle movement was energy dependent, since the addition of 2,4-dinitrophenol blocked the redistribution of vesicles completely. In addition, reduction of extracellular calcium concentration reversibly blocked vesicular movement as well. In conclusion, mammalian synaptic vesicles retain the cytoplasmic surface components necessary for translocation, sorting, and targeting to the proper locations by the native machinery of the squid giant synapse.
2,4-Dinitrophenol↗
Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.
A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.
Animals↗
Voltage-dependent calcium conductances in mammalian neurons. The P channel.
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Action Potentials↗