Acetylcholine receptor. Binding properties and ion permeability response after covalent attachment of the local anaesthetic quinacrine.

Membrane vesicles rich in nicotinic acetylcholine receptor prepared from Torpedo californica electric tissue have been irreversibly modified with quinacrine mustard, an alkylating derivative of the local anaesthetic quinacrine. The reaction blocked the ion channel regulated by the acetylcholine receptor. Acetylcholine still bound to the modified membrane vesicles with KD approx. 10(-8). The number of binding sites was reduced by up to 50%. Stopped-flow experiments showed that in contrast to what had been found with the reversibly binding quinacrine no fluorescence changes caused by energy transfer from the irradiated protein to the fluorescent local anaesthetic occurred after addition of agonist. This indicates that the conformational changes associated with the activation of the ion channel are blocked by the covalent reaction with quinacrine mustard. Analysis of the membrane vesicles by SDS-polyacrylamide gel electrophoresis showed that all polypeptide chains assumed to be part of the receptor complex had reacted with the mustard. Even small components, probably lipids, migrating with the dye front, showed fluorescence.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state

Quinacrine, like a typical local anaesthetic, blocks the response of Electrophorus electricus electroplaque in vivo in a non-competitive manner and enhances, in vitro, the affinity of the cholinergic receptor present in Torpedo marmorata membrane fragments for acetylcholine. The interaction of quinacrine with T. marmorata membrane fragments can be followed by differential fluorescence spectroscopy either upon direct illumination (λ_Ex = 350 nm) or by energy transfer from membrane proteins (λ_Ex = 290 nm). Carbamylcholine and most of the cholinergic ligands tested cause an increase of the light intensity emitted by membrane-bound quinacrine under conditions of direct excitation; all these effects are blocked by a preincubation of the membrane fragments with the α-toxin from Naja nigricollis. When quinacrine is excited by energy transfer, carbamylcholine, phenyltrimethylammonium and hexamethonium cause an increase of fluorescence but flaxedil, tetraethylammonium and the α-toxin give a much smaller fluorescence increase or none.Local anaesthetics like prilocaine or quotane cause a decrease of fluorescence intensity of membrane-bound quinacrine in both the presence and absence of carbamylcholine. Quantitative studies on quinacrine binding and fluorescence as a function of quinacrine concentration reveal at least two populations (saturable and non-saturable) of binding sites, the saturable one being identical or closely related to the specific site of action of local anaesthetics. It is concluded that binding of cholinergic ligands primarily increases the quantum yield of a fraction of bound quinacrine.The curves of variation of fluorescence intensity with agonist and antagonist concentrations determined under conditions of direct illumination, closely resemble the binding curves determined at equilibrium with radioactive ligands. Under these conditions quinacrine therefore enables us to determine the occupancy of the receptor site by cholinergic ligands. On the other hand, the change of quinacrine fluorescence observed by energy transfer, which takes place with some of the cholinergic ligands but not with others, and does not correlate with any variation of the intrinsic fluorescence of membrane proteins, most likely reflects a change of structure bearing a qualitative relationship to the pharmacological activity of the tested ligands.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.     

Time-dependent binding of paramagnetic and fluorescent hydrophobic ions to the acetylcholine receptor from Torpedo

In receptor-rich vesicles isolated from Torpedo, paramagnetic or fluorescent phosphonium ions bind to both the acetylcholine receptor (AcChR) and the receptor membrane. When added to receptor vesicles, two to three phosphoniums undergo a slow time-dependent binding to the AcChR. The presence of agonist increases the rate but not the extent of binding of the alkylphosphonium nitroxides. Approximately one phosphonium per receptor can be displaced by the addition of saturating concentrations of the high-affinity histrionicotoxin derivative isodihydrohistrionicotoxin or by the addition of phencyclidine or quinacrine mustard. In addition, preincubation of the receptor with these channel blockers prevents approximately one phosphonium from binding to the receptor. When a series of alkyltriphenylphosphonium ions was studied, it was found that the rate of phosphonium binding to the receptor decreased with increasing probe hydrophobicity. This appears to be a function of the partitioning of the probe between membrane and aqueous phases. The phosphonium ions used here promote desensitization of the receptor, as judged by the binding rate of the fluorescent agonist NBDA-C5-acylcholine or alpha-bungarotoxin. Preincubation of the receptor with isodihydrohistrionicotoxin virtually eliminates the phosphonium-mediated desensitization. The rates of the phosphonium-mediated desensitization also appear to be dependent upon the phase partitioning of the probe. These results strongly suggest that the binding sites for the phosphonium ion (and the high-affinity histrionicotoxin blocking site) are accessible only through the aqueous phase. The phosphonium binding and agonist-induced transitions observed here are not observed with a negative hydrophobic ion probe, or a negative surface amphiphile, indicating that modifications in membrane electrostatics do not contribute to the observed changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine-induced receptor-controlled ion flux investigated by flow quench techniques

Using a quench flow technique with membrane vesicles, the acetylcholine receptor-controlled transmembrane ion flux and the inactivation of the receptor with acetylcholine were measured in the msec time region. The ion flux was followed by influx of radioactive tracer ion and the inactivation was followed by an ion flux assay of receptor pre-incubated with ligand. The measurements covered a concentration range to complete saturation of the $\text{active}$ state of the receptor with ligand, and were consistent with a minimal model previously proposed on the basis of experiments with carbamylcholine. The ion translocation rate at saturation with acetylcholine is about twice that at saturation with carbamylcholine and this reflects a more favored channel opening equilibrium for acetylcholine.     

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.     

Interaction of quinacrine mustard with mononucleotides and polynucleotides

1. The interaction between quinacrine mustard and mononucleotides and polynucleotides was investigated by fluorimetry and absorbance spectrophotometry. 2. The fluorescence spectrum of quinacrine mustard is independent of the ionic strength and pH. The dependence of the quinacrine mustard fluorescence intensity on ionic strength, pH and anions is described. 3. The fluorescence intensity of quinacrine mustard was enhanced with the mononucleotide adenylic acid and polynucleotides such as poly(rA), poly(rU) and poly(rA,rU). 4. Quenching of the fluorescence intensity of quinacrine mustard occurred with the mononucleotide guanylic acid and with poly(rG) and poly(rC,rG). 5. The mononucleotide cytidylic acid or poly(rC) showed no effect on the fluorescence intensity of quinacrine mustard. 6. The interaction between the dye and native DNA species was also dependent on the presence of base-specific binding sites in the DNA. The higher the (G+C) content was in the native DNA tested the higher was the quenching effect on the fluorescence intensity of quinacrine mustard. 7. No interaction was found between the dye and methylated DNA. The binding between quinacrine mustard and apurinic DNA was confirmed to be in the phosphate groups of the purines.     

Change in the intrinsic fluorescence of acetylcholine receptor purified from Narke japonica upon binding with cholinergic ligands

Effects of various cholinergic ligands on the intrinsic fluorescence of acetylcholine receptor purified from the electric organ of Narke japonica were investigated. Binding with acetylcholine decreased the fluorescence by 7–8%, and that with carbamylcholine by 4–5% at 20 °C. Decamethonium and d-tubocurarine did not affect significantly the fluorescence intensity, while hexamethonium enhanced it. These changes were completely inhibited by preincubation of the receptor with α-bungarotoxin, which indicated that the observed intrinsic fluorescence change was due to the specific binding of each ligand. Data of the quenching experiment using iodide ion as an extrinsic quencher suggested the occurrence of the conformational change in the receptor upon binding with various cholinergic ligands. Considering these results together with those on intrinsic fluorescence change, conformational change provoked by binding with acetylcholine or carbamylcholine seems to differ from that provoked by binding with other cholinergic ligands examined.     

Quinacrine binds to the lipid-protein interface of the Torpedo acetylcholine receptor: a fluorescence study.

It has been argued both that there is a high affinity noncompetitive inhibitor binding site in the lumen of the acetylcholine receptor and that this lumen exists on the central axis of the receptor. Such a site would be expected to be 20-40 A from the membrane lipids. We tested whether, in fact, quinacrine, a potent fluorescent noncompetitive inhibitor, binds to such a site. We measured quenching of receptor-bound quinacrine fluorescence by fluorescence dipolar energy transfer to lipid probes, 5-(N-dodecanoylamino)eosin and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium, or by collision with paramagnetic lipid probes 2,2,6,6-tetramethylpiperidine-1-oxyl and 3-doxyl-17 beta-hydroxy-5 alpha-androstane (spin-labeled androstane). Initial control experiments established that in the presence of carbamylcholine, quinacrine binds to a phencyclidine-sensitive site on the Torpedo receptor with a Kd equal to 0.14 microM and with a quantum yield of 0.18. Fluorescence energy transfer from receptor-bound quinacrine had a magnitude consistent with quinacrine being less than 10 A from the lipid fluorescent probes. 2,2,6,6-Tetramethylpiperidine-1-oxyl and spin-labeled androstane were two to five times more effective at quenching receptor-bound quinacrine fluorescence than the fluorescence from membrane-partitioned 5-(dodecanoylamino)fluorescein. These results suggest that the quinacrine binding site is too close to the lipid domain to be in the lumen of the receptor, and therefore it is probably located on the outer surface of the membrane-spanning domain of the acetylcholine receptor.     

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel.

The reversible acetylcholine esterase inhibitor (-)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorata electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maelicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable-albeit only from other than the transmitter binding site(s).     

Reconstitution of a purified acetylcholine receptor

Dialysis of the purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organ results in a vesicular membrane system in which the receptor is embedded in the bilayer and oriented so that most of the neurotoxin-binding sites appear to be on the outer surface. The constituted vesicles are chemically excitable by acetylcholine and carbamylcholine, as measured by ²²Na⁺ efflux. The excitability is specifically blocked by the antagonist α-bungarotoxin. These results demonstrate that the purified reconstituted receptor system not only can specifically bind neurotransmitter but also can trigger ion translocation. It therefore has the properties necessary to effect postsynaptic depolarization in vivo.     

Actions of phencyclidine and its thienylpyrrolidine analogue on synaptic transmission and axonal conduction in the central nervous system of the cockroach Periplaneta americana

The effects of phencyclidine (PCP) and its thienylpyrrolidine analogue (TCPY) were tested on conduction processes in the isolated axon of giant interneurone 2 (GI 2) of the cockroach Periplaneta americana and on binding of [³H]PCP and [¹²⁵I]α-bungarotoxin to membranes from Periplaneta brain and nerve cord. Their actions on synaptic transmission between cercal sensory neurones and GI 2, where acetylcholine is the likely neurotransmitter, were also examined. PCP suppressed both sodium and potassium currents in the axonal membrane at 5.0 × 10^?4 M. Block was reversible on rebathing the axon in normal saline. TCPY exerted similar effects on the axon, though at slightly higher concentrations. Excitatory postsynaptic potentials (EPSPs) recorded from GI 2 in response to electrical stimulation of cercal nerve XI were progressively blocked by 5.0 × 10^?4 M PCP following a brief initial enhancement (?10%) of EPSP amplitude. The depolarizing response of GI 2 to ionophoretically applied acetylcholine was also blocked at this concentration, indicating a postsynaptic action of PCP at the acetylcholine receptor-ion channel of GI 2. TCPY also blocked synaptic transmission at synapses between cercal afferents and GI 2, but, in contrast to the actions of PCP, EPSP block was accompanied by depolarization. PCP and TCPY inhibited [³H]PCP binding to nerve cord and brain membranes with multiple affinities, suggesting multiple molecular targets. They also modified aspects of the kinetics of [¹²⁵I]α-bungarotoxin binding to the nicotinic acetylcholine receptor in these membranes and enhanced conversion of the receptor to the high affinity desensitized state. At higher concentrations they also inhibited [¹²⁵I]α-bungarotoxin binding. PCP was more potent than TCPY in inhibiting [³H]PCP binding but less potent on [¹²⁵I]α-bungarotoxin binding. Thus PCP and TCPY, which are structurally very similar, interact with several molecular targets in insect neuronal membranes, including sodium and potassium channels and acetylcholine receptors.     

Independent sites of low and high affinity for agonists on Torpedocalifornica acetylcholine receptor

It is demonstrated that two classes of binding site for acetylcholine are present on $\text{Torpedo}\text{californica}$ 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.     

A second pathway of activation of the Torpedo acetylcholine receptor channel.

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (-) physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, alpha-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor. Antibodies FK1 and benzoquinonium antagonize channel activation (and binding) of eserine, suggesting that the eserine binding site(s) is separate from, but adjacent to, the acetylcholine binding site at the receptor. In addition to the channel activating site(s) with an affinity of binding in the 50 microM range, there exists a further class of low-affinity (Kd approximately mM) sites from which eserine acts as a direct blocker of the acetylcholine-activated channel. Our results suggest the existence of a second pathway of activation of the nAChR channel.     

THE BINDING OF ACETYLCHOLINE TO SYNAPTIC VESICLES FROM TORPEDO CALIFORNICA ELECTROPLAX

Abstract— The in vitro uptake of exogenous acetylcholine by isolated presynaptic vesicles has been demonstrated in a new system. A preparation of vesicles from Torpedo californica electroplax was developed in which acetylcholinesterase and acetylcholine receptor activity were blocked. The vesicles bound acetylcholine with K_d 1.58 μM, the maximum amount bound being 26 pmol per g of original tissue, or 52 molecules per vesicle. Nicotinic drugs blocked binding, but muscarinic and noncholinergic drugs did not. The relative potency of nicotinic drugs differed greatly from their potency on Torpedo receptor. Sephadex chromatography showed that 26% of the binding was irreversible. The relationship of the binding to acetylcholine uptake and storage was discussed.     

Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes

Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.     

A monoclonal antibody interfering with binding and response of the acetylcholine receptor

Employing a monoclonal antibody raised against the receptor protein, we have probed the mechanism of ligand interaction of the nicotinic acetylcholine receptor from Torpedo marmorata. Antibody WF6 specifically binds to alpha-subunits of the receptor with a stoichiometry of one molecule per receptor monomer. At saturating concentrations, WF6 blocks half of the binding sites for acetylcholine, all of the binding sites for alpha-neurotoxins, and none of the binding sites for representative cholinergic antagonists (with the exception of alpha-toxins) at the receptor. In the presence of saturating concentrations of antibody WF6, acetylcholine (or its agonists) cannot induce T1+ influx into Torpedo membrane vesicles. Rapid oversaturation of the receptor by agonist also cannot overcome this blockade of channel gating. The observed competition patterns of WF6 and representative cholinergic ligands with the receptor are evidence for separate binding sites for groups of ligands and for a network of allosterically linked effector regions at the receptor. The blockade by saturating concentrations of WF6 of the agonist-induced channel gating supports the conclusion that two molecules of agonist are required to activate the receptor-integral ion channel.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor function were studied. At 1 × 10^?4M, the drug blocked 85% of the carbamylcholine-induced cation influx into liposomes, but left 90% of the αbungarotoxin binding intact. In addition, amantadine was shown to be a non-competitive inhibitor of membrane bound acetylcholinesterase. These experiments are relevant to the mechanism of action of amantadine at the motor end plate, where it produces electrophysiological changes compatible with an inhibition of cholinergic agonist mediated ion flux.