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1.
Jean-Pierre Changeux Thomas Podleski Jean-Claude Meunier 《The Journal of general physiology》1969,54(1):225-244
Several properties of the enzyme acetylcholinesterase (AChE) isolated in vitro are compared with those of the membrane receptor(s) of acetylcholine expressed by the in vivo electrical response of the electroplax membrane. AChE strongly binds in vitro effectors of the electroplax: agonists e.g., decamethonium or antagonists, e.g., d-tubocurarine and flaxedil. It also reacts covalently with an affinity labeling reagent of the acetylcholine receptor site(s) in vivo (TDF). Two classes of sites on AChE molecule account for the binding of these quaternary nitrogen containing compounds: (1) the anionic site of the active center and (2) noncatalytic "peripheral anionic centers" located outside the active center. A disulfide bond breaking agent, dithiothreitol (DTT) alters in a parallel manner the reaction of AChE and the excitable membrane of the electroplax to TDF. The irreversibility of TDF action is lost in both cases, after exposure to DTT. Both AChE and the acetylcholine receptor thus contain disulfide bonds—they are closely related but not necessarily identical proteins. 相似文献
2.
Arthur Karlin 《The Journal of general physiology》1969,54(1):245-264
The receptor for acetylcholine in the subsynaptic membrane of the electroplax of Electrophorus electricus is a protein with a disulfide bond in the vicinity of the active site. This disulfide can be reduced and reoxidized with concomitant inhibition and restoration of the response to acetylcholine and other monoquaternary ammonium-depolarizing agents. Conversely, the bisquaternary hexamethonium, normally a competitive inhibitor, causes depolarization, and the activity of decamethonium is increased following reduction of the disulfide. The reduced receptor can be alkylated by various maleimide derivatives and is then no longer reoxidizable. Some quaternary ammonium maleimide derivatives act as affinity labels of the reduced receptor, alkylating it at a rate three orders of magnitude faster then do uncharged maleimide derivatives. Other types of potential affinity labels also react only with the reduced receptor and the resulting covalently attached quaternary ammonium moieties interact with the active site, strongly activating the receptor. These results suggest a model for the active site and its transitions in which an activator such as acetylcholine bridges between a negative subsite and a hydrophobic subsite in the vicinity of the disulfide, causing an altered conformation around the negative subsite and a decrasee of a few angstroms in the distance between the two subsites. 相似文献
3.
Summary Exposure of frog sartorius muscle to the disulfide bond reducing agent, dithiothreitol, caused a decrease in the apparent affinity of their acetylcholine receptors for some agonists. Exposure to the oxidizing agent K3Fe(CN)6 reversed this effect. After reduction, the response to agonists was irreversibly blocked by exposure of the muscles to N-ethyl maleimide or 4-(N-maleimido)phenyltrimethylammonium. However, contrary to what had been observed with electroplax, the blocking reaction did not occur at the acetylcholine binding site of the receptor.This work is taken from a thesis presented by J. M. Lindstrom in partial fulfillment of the requirements for a Ph. D. degree in Biology, University of California, June 1971. 相似文献
4.
Lesley Harrington 《The Journal of general physiology》1973,62(1):58-76
The motor endplate of frog sartorius muscle was voltage clamped and the peak current to different concentrations of acetylcholine and carbachol applied in the perfusing fluid was measured. Perfusing fluid was hypertonic in order to suppress contractions. Current responses were smooth and reached a peak value within 2–5 s. The dose-response curve was usually linear even with concentrations of 10-2 M acetylcholine, indicating that the conductance change was probably proportional to the concentration of acetylcholine or carbachol. With high concentrations nonlinearity sometimes appeared but in these cases the fast onset of desensitization appeared to be preventing the current response from reaching its expected peak amplitude. When the depolarization produced by acetylcholine in a non-voltage-clamped endplate was measured the dose-response curve was hyperbolic. This relationship was imposed by the electrical properties of the endplate membrane and its surrounding sarcolemma, and could be predicted if the input resistance of the fiber was known. Experiments were also done on slow muscle fibers. Depolarizing analogues of acetylcholine had similar effects to acetylcholine. d-Tubocurarine reduced the proportionality constant between concentration of acetylcholine and conductance change, and this resulted in a parallel shift of the log-concentration depolarization curve. A linear dose-response curve was unexpected within the context of current theories of drug action. 相似文献
5.
The effects of nicotinic and muscarinic mimetics and lytics on spontaneous quantal transmitter secretion from the motor nerve endings were investigated during experiments on theRana temporaria sartorius muscle. Acetylcholine and carbachol reduced the frequency of miniature endplate potentials both in a normal ionic medium and in one with potassium ion concentration raised to 10 mM. Similar effects were produced by nicotinic agonists, namely nicotine, tetramethylammonium, and suberyldicholine, whereas muscarinic mimetics — methylfurmetide, oxotremorine, and F-2268 (L- and D-stereoisomers) — did not affect transmitter release. Neither d-tubocurarine, benzohexonium, nor atropine abolished the presynaptic effects of carbachol and acetylcholine. It is concluded that nicotinic cholinoreceptors are present at the frog motor nerve endings which modify spontaneous transmitter release and differ in their pharmacological properties from recognized N-cholinoreceptors of the motor and autonomic systems of the higher vertebrates.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 586–593, September–October, 1986. 相似文献
6.
The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system 总被引:2,自引:0,他引:2
Perhydrohistrionicotoxin at micromolar concentrations blocked the nicotine-evoked transmitter release from perfused striatal (dopaminergic) and hippocampal (cholinergic) nerve terminals. Perhydrohistrionicotoxin failed to compete with [3H]nicotine for its high-affinity binding site in rat brain, suggesting that the action of this toxin on central nicotinic receptors is noncompetitive. From the dose-response curve, 50% inhibition of nicotine-evoked striatal dopamine release occurred at 5 microM perhydrohistrionicotoxin, a value similar to that obtained in frog sartorius muscle and Electrophorus electroplax. This close agreement may suggest that the ionic channel of the presynaptic nicotinic acetylcholine receptor of brain neurons has similar properties to those of the peripheral receptor. 相似文献
7.
C Bravo J A Sobrino J Ortiz M D Comas J A García-Baro T del Riego 《Revista Espanola de Fisiología》1985,41(1):5-10
The effect that bath application of sulphydryl reagents (SR) exerts on frog sartorius motor endplate sensitivity to iontophoretically applied carbachol (CCh) has been studied. Sensitivity to CCh is expressed as the ratio of the CCh potential (mV) to the nanocoulombs delivered by the iontophoretic pulse and has been determined before and after addition of SR to the bath. Two groups of SR have been tested: oxidizing reagents, o-iodosobenzoate and reducing agents, dithiothreitol (DTT). CCh was applied iontophoretically by means of a microelectrophoretic programmer with constant current source. Exposure of the muscle to 1 mM DTT in a bath pH range of 7-8 for 2 to 85 min showed no significant differences in endplate sensitivity to CCh before and after addition of the reducing agent. o-Iodosobenzoate at a 1 mM bath concentration (pH 7) for 2 to 19 min strongly decreases endplate sensitivity to CCh. The statistical methods used were Wilcoxon rank tests and linear regression. Since previous studies have shown that oxidizing and reducing SR evoke depolarizations when applied iontophoretically at the endplate region, these results suggest that activation of the receptor is achieved only when SR are delivered iontophoretically, and that discrepancies observed can be attributed mainly to the different techniques of drug application. 相似文献
8.
Elena B. Pasquale Jayant B. Udgaonkar George P. Hess 《The Journal of membrane biology》1986,93(2):195-204
Summary Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax ofElectrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements withElectrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ ofE. electricus. 相似文献
9.
George B. Zavoico Carol Comerci Emily Subers John J. Egan Chi-Kuang Huang Maurice B. Feinstein Henry Smilowitz 《生物化学与生物物理学报:生物膜》1984,770(2)
The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits. 相似文献
10.
Robert N. Cox Masataka Kawai Arthur Karlin Philip W. Brandt 《The Journal of membrane biology》1979,51(2):145-159
Summary The depolarization that develops after covalent attachment of trimethylammonium benzoyl to the dithiothreitol-reduced frog sartorius acetylcholine receptor is accompanied by a small increase in voltage fluctuations. The amplitude of the elementary voltage event produced by the covalently attached activator is about 0.04 V, almost an order of magnitude below the acetylcholine shot-effect amplitude in the control preparation, and about one-fourth the acetylcholine shot amplitude after disulfide-bond reduction. Spectral density plots of trimethylammonium-benzoyl noise can be analyzed in terms of two relaxation rates that bracket the single rate observed in response to acetylcholine. 相似文献
11.
The ability of various organic cations to depolarize the ACh-activated endplate membrane in the absence of Na ions was examined on frog sartorius muscle by measuring the endplate potential on the muscle surface with the moving electrode technique. The ACh-activated endplate membrane was very permeable to ammonium and its methyl and hydroxy derivatives, and moderately permeable to guanidine derivatives and Tris (hydroxymethyl) aminomethane. The permeability of alkylol derivatives of ammonium diminished progressively with increase in molecular size. The present results suggested that the endplate ionic channels can be represented by a pore of about 6.4 Å in diameter. 相似文献
12.
G. G. Schofield J. E. Warnick E. X. Albuquerque 《Cellular and molecular neurobiology》1981,1(2):209-230
The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate of rise and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration. 相似文献
13.
Permeability of the endplate membrane activated by acetylcholine to some organic cations. 总被引:11,自引:0,他引:11
The ability of various organic cations to depolarize the ACh-activated endplate membrane in the absence of Na ions was examined on frog sartorius muscle by measuring the endplate potential on the muscle surface with the moving electrode technique. The ACh-activated endplate membrane was very permeable to ammonium and its methyl and hydroxy derivatives, and moderately permeable to guanidine derivatives and Tris (hydroxymethyl) aminomethane. The permeability of alkylol derivatives of ammonium diminished progressively with increase in molecular size. The present results suggested that the endplate ionic channels can be represented by a pore of about 6.4 A in diameter. 相似文献
14.
George B. Zavoico Carol Comerci Emily Subers John J. Egan Chi-Kuang Huang Maurice B. Feinstein Henry Smilowitz 《生物化学与生物物理学报:生物膜》1984,770(2):225-229
The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits. 相似文献
15.
Experiments were performed on chronically denervated frog sartorius muscles to determine the permeability of the acetylcholine-activated channels to organic cations. The membrane voltage response to bath-applied acetylcholine was measured with the moving electrode when the muscles were bathed in normal Ringer and in Ringer in which all of the Na+ had been replaced with an organic cation. The magnitude of the maximum voltage response was used to estimate the permeability of the channel to the organic cation. These results were compared with those which have been reported for innervated frog sartorius muscles (Maeno, Edwards, and Anraku, 1977). It is concluded that the permeability to a wide range of organic cations is virtually identical for the extrajunctional channels which develop following denervation and the channels which are localized at the junctional region of innervated muscles. 相似文献
16.
S M Dunn B M Conti-Tronconi M A Raftery 《Biochemical and biophysical research communications》1986,139(2):830-837
Torpedo acetylcholine receptor (AcChR) exists predominantly as dimers, formed by two monomers held together by a disulfide bridge(s). The dimers are easily cleaved to monomers by reducing agents. 2-mercaptoethanesulfonic acid is shown to be a membrane-impermeant reducing agent which cleaves receptor dimers when it is present only on the outside of intact membrane vesicles. There is no increase in the extent of cleavage when 2-mercaptoethanesulfonic acid is also loaded inside the vesicles. Therefore the disulfide bond(s) involved in the dimerization of the Torpedo acetylcholine receptor is (are) formed by cysteine residues which are exposed on the extracellular side of the membrane. 相似文献
17.
B Katz R Miledi 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1981,212(1186):131-137
Previous experiments have indicated that there is a continuous leakage of acetylcholine (ACh) from resting motor nerve terminals which can produce a small depolarization in anti-esterase treated endplates (Katz & Miledi 1977; Vyskocil & Illés 1978). This leakage might be expected to be intensified during the presynaptic action potential and also lead to a very small non-quantal endplate response. This hypothesis was examined, in frog and mammalian endplates, by stimulating the motor nerve in a calcium-deprived medium and recording the summated average response to several hundred stimuli. The result was completely negative; no trace of a non-quantal endplate potential was ever observed, with the limit of detection being always less than 10 microV, and sometimes as low as 2 microV. These experiments suggest that the leakage of ACh either does not originate predominantly from the synaptic region of the axon terminal, or that it occurs by a mechanism that is not directly influenced by the membrane potential. 相似文献
18.
Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, Km value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 108 acetylcholine molecules in 1 msec. Since it is estimated that 108 cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 105 . From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate. 相似文献
19.
R A Giniatullin R N Khazipov 《Biulleten' eksperimental'no? biologii i meditsiny》1989,108(12):654-657
The possibility of desensitization (DS) development under the rhythmic activity and during the generation of the single response to acetylcholine was studied in the frog sartorius muscle by voltage-clamp technique. It was revealed, that under conditions promoting DS (hyperpolarization, muscle warming, rise of calcium concentration as well as treatment by the DS--potentiating agent--proadifen) decrease of the postsynaptic membrane sensitivity to the acetylcholine can develop after few multiquantal end-plate currents--and when acetylcholinesterase is inhibited--during the response to a single quantum of acetylcholine. 相似文献
20.
Anson Cheng Marco Morsch Yui Murata Nazanin Ghazanfari Stephen W. Reddel William D. Phillips 《PloS one》2013,8(7)
Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm2 and nerve terminal synaptophysin 168±14 µm2 (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm2 area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates. 相似文献