Affinity For Acetylcholine Research Articles

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick.

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.

Further study of the correlation between Na-pump activity and membrane chemosensitivity.

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.

High-affinity binding of [3H]acetylcholine to muscarinic cholinergic receptors

High-affinity binding of [3H]acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the [3H]acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity [3H]acetylcholine binding sites to total muscarinic binding sites labeled by [3H]quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, [3H] acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that [3H]acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

Cooling-induced supersensitivity to acetylcholine in the isolated airway smooth muscle of the rat.

Isolated tracheal and bronchial strip-chain preparations of the rat were used to study the effect of temperature on electrically or acetylcholine-induced contraction. The preparations were suspended in the organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of bath temperature from 37 degrees C to 20 degrees C (cooling) had no effect on basal tone but augmented the contractile responses of the trachea and bronchus caused by stimulation of intramural cholinergic nerves (0.5-5 Hz) or acetylcholine (3 mumol/l-0.3 mmol/l). Cooling-induced augmentation of the contractile response to acetylcholine was not affected by pretreatment of the tissue with physostigmine (0.1 mumol/l) or tetrodotoxin (0.3 mumol/l). The affinity of acetylcholine for the tracheal muscarinic receptors at 20 degrees C, determined from its dissociation constant (KA), was not significantly different from that at 37 degrees C. On the other hand, acetylcholine-induced contraction of trachea which was incubated with isosmotic K+- rich Krebs solution and with Ca-free, EGTA (0.1 mmol/l) containing Krebs solution were both augmented at 20 degrees C. Caffeine or vanadate, each at a lower concentration than the threshold for causing contraction by itself, augmented the contractile responses of the trachea to acetylcholine (1 mumol/l-0.3 mmol/l). These potentiating effects of caffeine and vanadate were greater at 20 degrees C then 37 degrees C. From these observations, it is concluded that increased responsiveness of the rat airway smooth muscle to acetylcholine with lowered temperature may involve the acceleration of Ca release from intracellular storage sites, inhibition of Ca extrusion from the cell and or the inhibition of Ca reuptake by intracellular storage sites.

Effect of adenosine triphosphate on the sensitivity of the nicotinic acetylcholine-receptor in the bullfrog sympathetic ganglion cell.

The effects of adenosine triphosphate (ATP) and related compounds on the sensitivity of the nicotinic acetylcholine (ACh)-receptor of bullfrog sympathetic ganglion cells were analysed electro-physiologically. ATP in concentrations between 0.05 and 2 mM increased the amplitudes of the potentials and currents induced by ACh, and carbachol-induced currents. Compared with ATP, ADP was less potent in producing augmentation of the carbachol-induced current by one order of magnitude. AMP, cyclic AMP and adenosine had no appreciable effect. Analysis of this ATP effect, based on Michaelis-Menten type kinetics, revealed that ATP increased the maximum response (Vmax) of the dose-response curve of ACh currents without an appreciable effect on the affinity (Km) of ACh for its receptor. It is suggested that ATP increased the receptor sensitivity by acting on an allosteric site of the nicotinic ACh receptor-ionic channel complex which, thus, may be linked to an ATP receptor, probably of the P2-receptor type (Burnstock, 1981).

Agonist mediated conformational changes of solubilized calf forebrain muscarinic acetylcholine receptors

Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [ 3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85±5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation.

Effects of pancuronium and hexamethonium on paraoxon-induced twitch potentiation and antidromic firing in rat phrenic nerve diaphragm preparations.

The actions of pancuronium, a selective antagonist of acetylcholine (ACh) at nicotinic cholinoceptors at motor endplates, and hexamethonium, a selective antagonist of ACh at nicotinic cholinoceptors in autonomic ganglia, have been studied in rat phrenic nerve diaphragm preparations. The effects on paraoxon-induced twitch potentiation and antidromic firing (ADF) in the phrenic nerve, were compared with the effects on normal twitch tension and intracellularly recorded miniature endplate potentials (m.e.p.ps) and endplate potentials (e.p.ps.) In preparations exposed to paraoxon, pancuronium was found to be approximately 10 times more effective in reducing the potentiated component of the twitch than the component which corresponded to the pre-paraoxon twitch. A similar result was obtained with hexamethonium. Pancuronium and hexamethonium, in concentrations which reduced paraoxon-induced twitch potentiation but had no effect on the twitch tension of preparations not treated with paraoxon, reduced paraoxon-induced ADF. The lowest concentrations of pancuronium and hexamethonium required for this also reduced the amplitude of m.e.p.ps and e.p.ps. Dithiothreitol, a disulphide bond reducing agent which reduces the affinity of ACh for nicotinic cholinoceptors, enhanced the potency of pancuronium 2 to 3 fold. The same also applied for hexamethonium. It is concluded that the experiments failed to provide evidence for an action of ACh on prejunctional nicotinic cholinoceptors of the ganglionic-type being involved in the initiation by paraoxon of twitch potentiation and ADF. Furthermore, the results obtained can be explained by pancuronium and hexamethonium reducing the action of ACh at the postjunctional membrane.

Substance P modulates the sensitivity of the nicotinic receptor in amphibian cholinergic transmission.

The effect of substance P on the sensitivity of nicotinic acetylcholine (ACh) receptors of bullfrog sympathetic ganglion cells and frog skeletal muscle endplate was examined electrophysiologically. The amplitude of ACh-induced postsynaptic potential (ACh potential) and current (ACh current) were reversibly and dose-dependently reduced by substance P at low concentrations (0.42-42 microM). The mean amplitude of the miniature endplate potential (m.e.p.p.) was also reduced by substance P (4.2 microM). Substance P (4.2 microM) shifted the S-shaped dose-response curve of the ACh current downward. A Lineweaver-Burk plot constructed from the dose-response curve revealed that substance P depressed the maximum response (Vmax) without changing the apparent affinity (Km) of ACh for the receptor. Substance P (0.42-42 microM) did not alter the reversal potential of the ACh current of the endplate. The half-decay time of endplate current (e.p.c.) and its voltage-dependency were not altered by substance P in these concentrations. The depression of the ACh current by substance P may not be due to a blockade of the opened channel which has been activated by the preceding combination of ACh with the receptor. These results suggest that substance P suppresses the sensitivity of nicotinic ACh-receptors of the sympathetic ganglion cell and skeletal muscle endplate, acting on a certain allosteric site but not the recognition site of ACh in the receptor-ionic channel complex.

The relationship between stimulus-induced antidromic firing and twitch potentiation produced by paraoxon in rat phrenic nerve-diaphragm preparations.

The relationship between stimulus-induced repetitive antidromic firing (ADF) in the motor nerve and twitch potentiation produced by the organophosphate anticholinesterase paraoxon, has been investigated in rat diaphragm preparations. Little or no ADF was produced by paraoxon in preparations bathed in a Tyrode solution containing 1 mM calcium and 1 mM magnesium ions although the preparations showed marked twitch potentiation. Increases in the calcium:magnesium ion ratio produced a ratio-dependent increase in the ADF but had no consistent effect on peak twitch potentiation. Dithiothreitol, a disulphide bond reducing agent which decreases the affinity of acetylcholine for nicotinic cholinoceptors, abolished ADF but only modified the time course of twitch potentiation. Dithiothreitol, a disulphide bond reducing agent which decreases the affinity of acetylcholine for nicotinic cholinoceptors, abolished ADF but only modified the time course of twitch potentiation. It is concluded that the initiation of ADF is a consequence of the prolonged action of acetylcholine within the synaptic cleft following inhibition of acetylcholinesterase, and that ADF is not the only mechanism by which twitch potentiation can be produced.

On the mechanism of calcium actions on the acetylcholine receptor-channel complex at the frog end-plate membrane.

The mechanism of depressant effects of Ca2+ on the acetylcholine (ACh)-activated ion channel conductance was studied in frog muscle fibers. Increasing the extracellular Ca2+ ( (Ca2+)o) suppressed the increase in the end-plate conductance induced by iontophoretic application of ACh and shifted the reversal potential for a neurally-induced end-plate current to a more negative value. The single channel conductance obtained by current fluctuation analysis was reduced at a high (Ca2+)o. The decay phase of a neurally-induced end-plate current was slightly prolonged at a high (Ca2+)o (18 mM), while that of miniature end-plate current and the open time of the end-plate channel measured by noise analysis remained unchanged under the same condition. The Hill coefficient and apparent dissociation constant as measured by quantitative iontophoresis of ACh and comparison of the end-plate response with a theoretical model (DREYER and PEPER, 1975) were not affected by raising (Ca2+)o, while the maximum conductance increase was suppressed. These results suggest that the depressant effect of Ca2+ on the end-plate channel is not due to the effects on surface charges of the membrane, affinity of ACh to the ACh-receptor or binding to intrachannel sites, but presumably arises from the effect on another site involved in the regulation of ion permeation of the receptor-channel complex.

Twitch potentiation by organophosphate anticholinesterases in rat phrenic nerve diaphragm preparations.

1 Twitch potentiation produced by anticholinesterases has been variously attributed to the prolonged postjunctional action of acetylcholine (ACh), a prejunctional action of ACh involving the initiation of antidromic firing (ADF) in the nerve or a direct action of the anticholinesterases on nerve terminals initiating ADF. 2 The organophosphate anticholinesterases, paraoxon (diethyl-4-nitrophenylphosphate) and DFP (diisopropyl fluorophosphate), when applied to rat isolated diaphragm preparations for 30 min, produced twitch potentiation which subsequently declined. 3 The rates of onset and decline of twitch potentiation were directly related to the concentration of the organophosphates and the reversibility of their effects was in line with the reactivation of the phosphorylated enzymes formed by them, whether reactivation was spontaneous or induced by the oxime, N,N'-trimethylene-1, 3-bis(pyridinium-4-aldoxime). 4 Reducing the output of ACh from nerve terminals (by reducing the ratio of calcium: magnesium ions in the bathing solution) or reducing the affinity of ACh for the nicotinic cholinoceptor (using the disulphide bond reducing agent, dithiothreitol) produced the same effects as did lowering the concentration of the organophosphates. 5 It is concluded that the twitch potentiation produced by paraoxon and DFP, and its failure to be maintained when the higher concentrations of the organophosphates were used, were the direct result of the excess of ACh in the synaptic cleft, following inhibition of acetylcholinesterase.

A comparison of the muscarinic cholinoceptors and benzodiazepine receptors of guinea-pig brain and rat brain

Some properties of muscarinic cholinoceptors and benzodiazepine receptors in selected brain regions of guinea-pigs and rats were compared under identical experimental conditions. The regions investigated were striatum, hippocampus and pons-medulla, and the properties examined were the concentrations of receptors; apparent dissociation constants of the ligands [ 3H]quinuclidinyl benzilate (for muscarinic receptors) and [ 3H]flunitrazepam (for benzodiazepine receptors); Hill coefficients for the interactions of the antagonist atropine and the agonist acetylcholine with the muscarinic receptors; the affinities of these compounds for the muscarinic receptors; and the effects of chronic administration of an organophosphate cholinesterase inhibitor (di-isopropylfluorophosphate) on the concentrations of receptors. Rat striatal and hippocampal muscarinic receptors were found to have a slightly higher affinity for acetylcholine than the corresponding guinea-pig receptors. Administration of di-isopropylfluorophosphate reduced the concentration of muscarinic receptors in rat brain by 30%, but had no significant effect on the concentration of receptors in guinea-pig brain. In all other aspects, the properties of the brain receptors of the two species were very similar. For both species, the affinities of the muscarinic receptors for acetylcholine were higher in the pons-medulla than in the striatum and hippocampus. This was found to be the result of differences in the values of the association constants of the high- and low-affinity states of the receptors, rather than because of varying proportions of two states which have the same association constant in all regions. The insensitivity of guinea-pig brain muscarinic receptors to chronic administration of an organophosphate confirms the results of a previous study on the guinea-pig alone, and makes this system unique. Many other studies on various species have all indicated that prolonged activation of a receptor by an agonist (caused in the present work by inactivation of acetyl-cholinesterase) leads to a decrease in the concentration of the receptor.

Independent sites of low and high affinity for agonists on [formula omitted] acetylcholine receptor

It is demonstrated that two classes of binding site for acetylcholine are present on Torpedo californica acetylcholine receptor. One class is the well documented site on each of the two subunits of 40,000 daltons, which can be covalently modified by bromocetylcholine. Both in the absence and in the presence of bromoacetylcholine another binding site is shown to exist by virtue of acetylcholine dependent fluorescence changes in the receptor covalently modified by 4-[N-(iodoacetoxy)ethyl-N-methyl]-amino-7-Nitrobenz-2-oxa-1,3 diazole (IANBD). This site has a low affinity for acetylcholine (K d ∼ 80 μM) that corresponds closely with the known concentration dependence of acetylcholine mediated activation of this receptor and we conclude that it may represent a site of association that participates in channel opening in this system.

Mechanisms of acetylcholine receptor loss from the neuromuscular junction.

At the normal mammalian neuromuscular junction the half-life of the acetylcholine receptor (AChR) ranges from 6 to 13 days (estimates from seven different laboratories). Indirect evidence suggests that the internalized receptor is degraded by a lysosomal mechanism. We have now traced the fate of the AChR labelled in vivo with peroxidase-alpha-bungarotoxin. Segments of junctional folds bearing AChRs are internalized by endocytosis. The endocytosed vesicles are engulfed by tubules and larger vesicles which, by electron cytochemical criteria, represent secondary lysosomes. Pathological mechanisms increased AChR loss from the end-plate. These include destruction of junctional folds, formation of immature junctions with a few or no junctional folds, accelerated internalization of AChR, impaired membrane insertion of new AChR and, possibly decreased AChR synthesis. The common mechanism for destruction of the junctional folds is an altered subsynaptic ionic milieu, and especially focal calcium excess. This can be induced by antibody and complement, too frequent or prolonged openings of the acetylcholine (ACh)-induced ion channel, and other membrane defects. In acquired autoimmune myasthenia gravis there is (a) antibody-dependent complement-mediated lysis of the junctional folds, (b) accelerated internalization of AChR cross-linked by antibody and (c) decreased insertion of AChR into the postsynaptic membrane. The last mechanism is attributed to lack of membrane patches available for tight packing and secure anchoring of the receptor. In acute, but not in chronic, experimental autoimmune myasthenia gravis, and infrequently in human myasthenia gravis, macrophages destroy junctional folds opsonized by antibody and C3. In a recently recognized congenital syndrome attributed to a prolonged open time of the ACh-induced ion channel, and to a lesser extent in congenital end-plate acetylcholinesterase deficiency, AChR is lost with degradation of junctional folds. In other, less well-defined, congenital syndromes there is deficiency or abnormal function of AChR. This could arise from decreased synthesis or membrane insertion or accelerated degradation of AChR, or from a structurally abnormal AChR with reduced affinity for ACh or with a diminished conductance or open time of its ion channel.

Increase of acetylcholine-receptor sensitivity by adenosine triphosphate: a novel action of ATP on ACh-sensitivity.

1. The sensitivity of the nicotinic acetylcholine (ACh)-receptor, measured as the amplitude of ACh-current induced by iontophoretic application of ACh to the frog skeletal muscle endplate, was increased by the action of adenosine triphosphate (ATP). 2. This potentiation was not due to the effect of ATP on ACh-esterase, since the increase of the sensitivity could also be demonstrated by use of carbachol (CCh). 3. Kinetic analysis of the effect of ATP on the dose-response curve of CCh-current suggests that ATP increases the ACh-sensitivity by acting on the allosteric site of receptor-ionic channel complex without changing the affinity of ACh for its recognition site. 4. The equilibrium potential and the life-time of the endplate current (e.p.c.) are not altered by the presence of ATP. 5. These results suggest that ATP increases the ACh-sensitivity by increasing either the conductance of unit channels or the total number of available channels.

5-Hydroxytryptamine controls ACh-receptor sensitivity of bullfrog sympathetic ganglion cells

Experimental evidences showing that 5-hydroxytryptamine (5-HT) is directly interacting with nicotinic acetylcholine (ACh) receptors and thereby depresses the sensitivity of these receptors to ACh, are presented by making use of bullfrog sympathetic ganglion cells and frog skeletal muscle end-plates. It was suggested that 5-HT might decrease the affinity of ACh to nicotinic receptor sites, since the mode of 5-HT action was comparable to that of d-tubocurarine action.

An analysis of the action of a false transmitter at the neuromuscular junction.

1. The action of monoethylcholine (MECh) on neuromuscular transmission has been studied by electrophysiological methods. 2. End-plate potentials (e.p.p.s.) in curarized rat muscle were unaffected or slightly increased in amplitude by MECh (0-1-1 mM). Stimulation at 3 Hz for about 30 min in the presence of MECh caused a progressive decline in e.p.p. amplitude, and a shortening of the e.p.p. time course. These changes were reversed by addition of choline to the medium. Similar changes in amplitude, but no change in time course, occurred when the preparation was stimulated in the presence of hemicholinium or triethylcholine. 3. Extracellular recordings of miniature end-plate potentials in frog muscle showed that stimulation in the presence of MECh caused the time constant of the exponential decay of the m.e.p.p.s. to decrease by 42%. The amplitude of intracellular m.e.p.p.s. was reduced by 45%. These changes were maximal by the time about 3 X 10(5) quanta had been released. 4. Voltage clamp experiments in rat muscle in which miniature end-plate currents (m.e.p.c.s) were recorded showed that stimulation in the presence of MECh reduced the amplitude (by 33%) and the decay time constant (by 42%). 5. Analysis of end-plate current flucutations produced by local application of acetylcholine (ACh) and acetylmonoethycholine (AMECh) to voltage clamped rat end-plates showed that the amplitude of the elementary current events was the same for both compounds whereas the average channel lifetime was 44% shorter for AMECh than for ACh. 6. The voltage-sensitivity of the channel lifetime (measured from end-plate current fluctuations) was the same for ACh and AMECh. The voltage-sensitivity of the m.e.p.c. decay time constant was the same as that found from noise measurements. The shortened m.e.p.c.s. (false m.e.p.c.s.) occurring after stimulation in the presence of MECh also showed the same voltage-sensitivity. 7. Both normal and false m.e.p.c.s. were prolonged by neostigmine by almost the same factor; false m.e.p.c.s. were thus shorter than normal m.e.p.c.s. even when cholinesterase was inactivated. Experiments with progressive curarization of neostigmine-treated end-plates suggested that the fraction of transmitter molecules bound is smaller for false than for normal m.e.p.c.s. The difference implies that the false transmitter has one quarter of the affinity of ACh for the receptors. 8. It is concluded that stimulation in the presence of MECh gives rise to a false transmitter, presumably AMECh, which has a lower affinity for receptors than ACh, and gives rise to ionic channels with a shorter average lifetime than those activated by ACh.

Acetylcholine receptor oligomers from electroplax of Torpedo species.

Sedimentation in sucrose gradients of alpha-bungarotoxin-labeled crude and pure acetycholine receptor preparations from Torpedo californica showed two major oligomers. The molecular weights, corrected for the bound Triton X-100 by comparing sedimentation in H2O and in D2O, were 330 000 for the heavy (H) oligomer and 190 000 for the light (L) oligomer. Lesser peaks found in preparations of T. marmorata and purified preparations of T. californica with molecular weights of 500 000 (HH) and 80 000 (LL). These molecular weights are based upon the assumption of globularity, and may require adjustment if the assumption is wrong. The H and L peaks have similar drug sensitivities, but at pH 10 the L peak was stable whereas the H peak dissociated to components sedimenting as LL. Treatments with p-chloromercuribenzoate, which blocks acetylcholine binding partially without affecting alpha-bungarotoxin binding, had no effect upon the pattern of sedimentation. This and other evidence suggested that the heterogeneity of oligomers was unrelated to the heterogeneity of site affinities for acetylcholine and nicotinic drugs.

Interconversion between different states of affinity for acetylcholine of the cholinergic receptor protein from Torpedo marmorata.

In receptor-rich membrane fragments from Torpedo, acetylcholine binds, in the presence of 70 muM Tetram, to a homogeneous population of high-affinity sites with Kd = (3.4 +/- 0.8) x 10(08) M. Dissolution of these membrane fragments by sodium cholate causes a decrease of affinity associated with the appearance of medium-affinity (Kd approximately 10(-7) M) and low-affinity (Kd greater than or equal to 10(-6) M) sites. Dissolution by neutral detergents Triton X-100 or Emulphogene preserves the high affinity of the acetylcholine binding sites. In all the soluble states of the receptor protein, Ca2+ ions and local anaesthetics no longer enhance the affinity for acetylcholine. Elimination of sodium cholate by dilution leads to the reassociation of the receptor protein, the recovery of high-affinity sites and the control by Ca2+ ions and local anaesthetics. Purification by affinity chromatography of the receptor protein in Triton X-100 is accompanied by a conversion of a majority of the acetylcholine sites into their state of low affinity. High-affinity sites can no longer be recovered by detergent dilution from these low-affinity ones.

30] α-Bungarotoxin (and similar α-neurotoxins) and nicotinic acetylcholine receptors

Publisher Summary Chemical transmission of impulses from nerves to vertebrate skeletal muscles and electric tissues is mediated by the neurotransmitter acetylcholine (ACh). When an action potential in a nerve axon reaches and depolarizes a nerve terminal, it triggers a calcium-dependent process for the neurosecretion of ACh into the synaptic cleft. Roughly a million molecules of ACh appear for each nerve impulse. Some of these diffuse to the postsynaptic cell and react with recognition sites; these “receptors” on the outside of the postsynaptic cell membrane have high affinity and specificity for ACh. The ACh receptors of skeletal muscles were called “nicotinic” by Dale because they could be distinguished from others pharmacologically: they (like those of electric tissues) are activated by nicotine and antagonized competitively by d -tubocurarine.