Acetylcholine receptor: evidence for a regulatory binding site in investigations of suberyldicholine-induced transmembrane ion flux in Electrophorus electricus membrane vesicles

Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1 degree C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (86Rb+). JA, the rate coefficient for ion flux mediated by the active form of the receptor, and alpha, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 microM. At higher suberyldicholine concentrations (greater than 50 microM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, KR, is consistent with the results obtained over a 10 000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (KR = 500 microM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient alpha [alpha(max) = 5.7 s-1], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [JA(max) = 18.5 s-1] is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used.(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.     

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.     

Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

The issue of whether d-tubocurarine, the classical acetylcholine receptor inhibitor, inhibits the receptor by a competitive or noncompetitive mechanism has long been controversial. d-Tubocurarine, in this study, has been found to be both a competitive (KC = 120 nM) and a noncompetitive (KNC = 4 microM) inhibitor of receptor-mediated ion flux at zero transmembrane voltage in membrane vesicles prepared from Electrophorus electricus electroplax. A spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, was used to measure both the rate coefficient of ion flux prior to receptor inactivation (desensitization) and the rate coefficient of the rapid inactivation process. Inhibition by d-tubocurarine of the initial rate of ion flux decreased with increasing acetylcholine concentration, consistent with competitive inhibition, but the inhibition by micromolar concentrations of d-tubocurarine could not be overcome with saturating concentrations of acetylcholine, consistent with noncompetitive inhibition. A minimum mechanism is proposed in which d-tubocurarine competes for one of the two acetylcholine activating sites and also binds to a noncompetitive site. The present data do not distinguish between one or two competitive sites, although one successfully accounts for all of the data. By variation of the acetylcholine concentration, the two types of sites could be studied in isolation. Binding of d-tubocurarine to the noncompetitive site does not change the rate of rapid receptor inactivation, whereas binding of d-tubocurarine to the competitive site decreases the rate of rapid inactivation by displacing acetylcholine, in agreement with the observation that d-tubocurarine does not inactivate (desensitize) the E. electricus receptor by itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-organization effects induced by ion-conformational interaction in biomembrane channels

It is shown that phenomena of molecular self-organization can take place in biomembrane channels. A strong interaction between charged groups of channel-forming protein and ion flux passing through the channel causes bistable or steady limit cycle regimes. These regimes are possible only far from the equilibrium state when the ion flux exceeds a certain threshold value. A number of such synergetic models is reviewed. Each time, only the simplest (monostable) regime takes place near the zero value of the ion flux. Some experimental data may be explained by this approach.     

Effects of thio-group modifications of Torpedo californica acetylcholine receptor on ion flux activation and inactivation kinetics

The effects of thio-group modifications on the ion permeability control and ligand binding properties of the acetylcholine receptor were measured in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and soybean lipids (asolectin). A quench flow device was used to obtain subsecond time resolution for agonist-stimulated cation influx using carbamylcholine chloride (Carb) as the ligand and 86Rb+ as the cation. The effects of disulfide reduction with dithiothreitol (DTT), affinity alkylation with [4-(N-maleimido)benzyl]trimethylammonium ion and bromoacetylcholine, and nonspecific alkylation with N-ethylmaleimide and N-benzylmaleimide were examined. Activation, fast inactivation, and slow inactivation rates were measured on the chemically modified membranes. The flux results were compared with similar measurements on native membranes, and the role of vesicle size, heterogeneity, and influx time on ion flux results was analyzed. Major conclusions are that the binding sites that react with affinity labels are the same sites that mediate ligand-activated ion flux and that blockade of one of the two ligand binding sites is sufficient to block about 95% of the ion flux response. The main effect of DTT reduction is to shift the EC50 values for activation and slow inactivation to higher Carb concentrations, consistent with a decrease in binding affinity for Carb. The EC50 value for fast inactivation was not affected by DTT. However, the maximum rate of ion flux activation and the maximum rate of fast inactivation were decreased 2-fold after DTT treatment.     

Reconstitution of acetylcholine receptor function in lipid vesicles of defined composition

The effect of specific lipids on the functional properties of the acetylcholine receptor were examined in reconstituted membranes prepared from purified Torpedo californica acetylcholine receptor and various defined lipids. Cholesterol and negatively charged lipids greatly enhanced the ion influx response of the vesicles as measured by the effect of a receptor agonist on cation translocation across the vesicles. Part of the lipid-dependent effects could be attributed to alterations in the average size of the vesicles. All lipid mixtures used permitted complete incorporation of receptor and retention of ligand binding properties. Quantitative differences in ion flux properties suggest a modulating role for specific lipids in acetylcholine receptor function.     

Receptor stability and channel conversion in the subsynaptic membrane of the developing mammalian neuromuscular junction

Using electrophysiological and autoradiographic techniques, the gating properties and the metabolic stability of acetylcholine receptor-channel complexes were measured in the end-plate membrane of neonatal rat soleus muscle at various stages of postnatal development. Analysis of the decay time course of miniature end-plate current recordings suggests that a conversion of channel gating properties from a slowly relaxing to a rapidly relaxing type of end-plate channel, as found in the end-plate of adult fibers, occurs between day 8 and day 18 of postnatal development and can be described as a first-order process with a half-conversion time of ?3–4 days. Silver grain counts of autoradiograms of end-plates labeled with ¹²⁵I-α-bungarotoxin and subsequently maintained in organ culture for various times indicate that subsynaptic acetylcholine receptors have a metabolic half-life time ≥9 days, comparable to the value observed in adult fibers, already at the time of birth. This is taken as evidence that, during synaptogenesis, receptor and channel properties are controlled by different regulatory signals from the nerve terminal. A comparison of the time course of channel conversion and of receptor incorporation suggests that the postnatal change in end-plate channel properties is not the result of incorporation of a different form of receptor-channel complex.     

Interaction of a semirigid agonist with Torpedo acetylcholine receptor

The binding of the semirigid agonist [(3)H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (K(d) = 99 +/- 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor-ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC(50) = 31 +/- 5 microM) than acetylcholine but its maximum flux rate was 4-10-fold lower. This phenomenon has been studied further by single-channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 microM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 microM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor-ligand complex can adopt, thus favoring the lower conductance state.     

The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins.

In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain.

The activation and desensitization kinetics of the human alpha(1)-homooligomeric glycine receptor, which was transiently expressed in HEK 293 cells, were studied with a 100-microseconds time resolution to determine the rate and equilibrium constants of individual receptor reaction steps. Concentration jumps of the activating ligands glycine and beta-alanine were initiated by photolysis of caged, inactive precursors and were followed by neurotransmitter binding, receptor-channel opening, and receptor desensitization steps that were separated along the time axis. Analysis of the ligand concentration-dependence of these processes allows the determination of 1) the rate constants of glycine binding, k(+1) approximately 10(7) M(-1) s(-1), and dissociation, k(-1) = 1900 s(-1); 2) the rates of receptor-channel opening, k(op) = 2200 s(-1), and closing, k(cl) = 38 s(-1); 3) the receptor desensitization rate, alpha = 0.45 s(-1); 4) the number of occupied ligand binding sites necessary for receptor-channel activation and desensitization, n >/= 3; and 5) the maximum receptor-channel open probability, p(0) > 0.95. The kinetics of receptor-channel activation are insensitive to the transmembrane potential. A general model for glycine receptor activation explaining the experimental data consists of a sequential mechanism based on rapid ligand-binding steps preceding a rate-limiting receptor-channel opening reaction and slow receptor desensitization.     

Endogenous and exogenous proteolysis of the acetylcholine receptor from torpedo californica

Purified acetylcholine receptor reconstituted into liposomes catalyzes carbamylcholine-dependent ion flux [10]. An endogenous protease activated by Ca²⁺ gives rise to an acrylamide gel pattern of the receptor with the 40,000-dalton subunit apparently as the major component. Exogenous proteases nick the proteins so extensively that the acrylamide gel pattern reveals polypeptides of 20,000 daltons or less. In either case the receptor sediments at 9S, indicating that the polypeptide chains associated. Moreover, the nicked receptors bind α-bungarotoxin and catalyze carbamylcholine-dependent ion flux after reconstitution.     

Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.     

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: A minimal mechanism based on rate measurements in the millisecond to minute time region

The dependence of acetylcholine receptor-controlled transmembrane ion flux on carbamylcholine concentration was measured in the msec time region, using membrane vesicles and a quench flow technique. 4 Measurements were made: (1) transmembrane ion influx, (2) rate of inactivation of the receptor by carbamylcholine, (3) rate of recovery, and (4) ion influx mediated by “inactivated” receptor. The minimal model, based on the measurements, accounts for the time dependence of receptor-controlled ion flux over a 200-fold carbamylcholine concentration range. The maximum flux rate of 84 sec^?1 indicates that we have succeeded in measuring the receptor-controlled processes which give rise to electrical signals in cells.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

Ion channels and membrane potential in stimulus-secretion coupling in adrenal paraneurons

Cultured bovine adrenal medulla cells have been shown to contain several different ion channels (Na+, Ca2+, acetylcholine receptor regulated) whose activation leads to the secretion of catecholamines. The pharmacology of these ion channels and their interactions during secretion have been examined. The mechanisms of agonist-induced calcium influx are of particular interest since this is an early obligatory event during secretion from the adrenal medulla. Data obtained on catecholamine release and 45Ca2+ uptake indicate that both voltage-dependent and voltage-independent calcium influx mechanisms operate in cultured bovine adrenal medulla cells. The significance of these results in understanding the mechanism of action of the physiological stimulus acetylcholine (Ach) will be discussed. The alkaloid channel neurotoxins D-600, batrachotoxin, veratridine, and aconitine were shown to exert a noncompetitive inhibitory effect on Ach-induced ion flux in adrenal medulla cells, presumably through an interaction with the nicotinic receptor regulated channel. Lipid-soluble neurotoxins may interact with multiple ion channels in nerve and muscle membrane.     

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.     

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.