Apparent cooperative effects in acetylcholine receptor-mediated ion flux electroplax membrane preparations.

The kinetics of acetylcholine receptor-mediated flux of ²²sodium ions from microsacs has been measured in the presence of activators (carbamylcholine and decamethonium) and an inhibitor (d-tubocurarine) of neural transmission. The dependence of the first-order rate constant, k_obs, for ²²sodium ion efflux on either decamethonium or carbamylcholine concentration does not exhibit cooperativity. The apparent cooperativity observed by Kasai and Changeux in dose-response curves for ²²sodium flux from the same preparation is adequately accounted for by the contribution which efflux from non-excitable microsacs, the main component of the preparation, makes to the measurements. d-Tubocurarine was found to be a non-competitive inhibitor of decamethonium-activated ²²sodium efflux. The results of the kinetic measurements are in agreement with equilibrium measurements of the interaction of decamethonium with the same microsac preparation, i.e. adherence to a classic Langmuir binding isotherm and separate binding sites for activators and inhibitors of neural activity. The results indicate a direct relationship between ligand binding and receptor-mediated ion flux. How these two processes contribute to electrophysiological measurements is not apparent.     

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 gating events in tracer flux experiments. III. acetylcholine receptor-controlled Li+ efflux from sealed Torpedo marmorata membrane fragments

Filter assay measurements of Li+ efflux from acetylcholine receptor-containing vesicular Torpedo marmorata membrane fragments (microsacs) are presented. Techniques are introduced for: (a) inducing a complete emptying of the Li+ content of all microsacs containing one or more functionally intact receptors, and (b) for determining the distribution of internal volumes of the microsacs using filtration with membrane filters of different pore sizes. The flux amplitudes resulting for acetylcholine receptor-controlled Li+ efflux, when receptors are inhibited by alpha-bungarotoxin or inactivated by a neuroactivator-induced desensitization process, were measured. Amplitude analysis was used to determine characteristic parameters of the microsacs that may vary with the technique of preparation (e.g., the distribution in size and receptor content), as well as the mean single channel flux amplitude contribution (e-kt)infinity, which represents the mean reduction of the Li+ content of a microsac due to efflux from a single receptor-controlled channel closing due to inhibition or inactivation of the receptor. The ratio keff/ki was found to lie in the range 0.1 less than keff/ki less than 0.5, where keff and ki are, respectively, the rate constant for Li+-Na+ exchange flux and for the slow inactivation reaction mode of the acetylcholine receptor induced by carbamoylcholine at high concentrations.     

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.     

Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles

A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of ²²Na⁺ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist-induced uptake of ²²Na⁺ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists (‘desensitization’) can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles.     

Phencyclidine inhibition of the acetylcholine receptor: measurement of cation flux in a sympathetic neuronal cell line using 22Na+ and spectroscopic detection of Cs+

The site of action of phencyclidine, a powerful and increasingly abused drug, in sympathetic nerve cells has not previously been identified. Here it is demonstrated that phencyclidine is a powerful, noncompetitive inhibitor of the nicotinic acetylcholine receptor in a sympathetic nerve cell line, PC-12. In the presence of 1 mM carbamoylcholine the rate of the receptor-controlled influx of 22Na+ is reduced by a factor of 2 by 0.7 microM phencyclidine. Increasing concentrations of carbamoylcholine cannot reverse the inhibitory effect of the drug. Both the transmission of electrical signals between nerve cells and the secretion of catecholamines in the PC-12 cell line depend on the receptor-controlled ion flux. Thus phencyclidine interferes with at least two specific, physiologically important functions of these nerve cells. A new spectroscopic method has been developed to measure cation flux in cells. It is shown that this method can replace measurements of tracer ion flux.     

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)     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108-116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na+/Cl-/glycine cotransport system is discussed.     

Fluorescence measurements of anion transport by the GABAA receptor in reconstituted membrane preparations

A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4509-4513]. The GABAA receptor has been solubilized from bovine brain membranes and reconstituted into phospholipid vesicles. Influx of chloride or iodide into the vesicles has been measured in stopped-flow experiments by monitoring the fluorescence quench of an anion-sensitive fluorophore trapped within the vesicles. Muscimol, a GABAA receptor agonist, stimulated a rapid uptake of either chloride or iodide. Stimulation of chloride influx was dependent on the concentration of muscimol, and the midpoint of the dose-response curve occurred at approximately 0.3 microM. Agonist-stimulated uptake was enhanced by diazepam and blocked by desensitization and by the antagonists bicuculline and picrotoxin. These receptor-mediated effects are shown to be qualitatively similar to measurements of 36Cl- and 125I- efflux using synaptoneurosomes prepared from rat cerebral cortex. The advantages of the fluorescence method in terms of its improved time resolution, sensitivity, and suitability for quantitating GABAA receptor function are discussed.     

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin

In order to establish whether or not chloride ions behave as freely moving particles in “passive”, i.e. ouabain-and acetazolamide-treated, frog skin, tracer fluxes of ³⁶Cl⁻ have been measured while a voltage (generally +40 mV, serosal side positive) across the skin was applied. Ussing's flux ratio equation has been used as a criterion for this type of transport. One group of skin samples exhibited significant exchange diffusion phenomena. Most samples in a second group either behaved according to the flux ratio equation or showed significant and extreme exchange diffusion. From flux ratios obtained at two different voltages across various skin samples, showing extreme exchange diffusion, it appeared that the simple form of Kedem and Essig's law derived from irreversible thermodynamics, which is valid for homogeneous systems, does not apply to the type of exchange diffusion found. The system can, however, be described by a 1 : 1 exchange mechanism working in parallel with a diffusional pathway. The ratio exchange flux/observed efflux must then have a constant value (0.83) at the voltages applied, which implies that the exchange flux is voltage dependent. By comparison with iodide flux experiments as carried out by Ussing, it is shown that iodide exhibits the same type of exchange diffusion. A carrier, possibly responsibe for the observed behaviour, is described.     

Characterizing soil fertility by ion diffusive flux measurements

Summary The general acceptance of diffusion as a rate-limiting step in transferring ions to plant roots was considered in characterizing soil fertility. Soil ion diffusive flux measurements to an exchange resin were measured with two soil: resin cells, one with untreated soil, the other with the same soil fertilized with P, K, and Cl. The P, K, and Cl that diffused to the resin of each cell was extracted and determined. Using only these measurements and some simplifying assumptions of diffusion theory, calculations were made of the soil's P and K solution concentration, adsorbed concentration, a capacity factor, transmission factor, and quantity of fertilizer needed to attain any desired flux. Excellent to fair correlations existed between these calculated values and traditionally measured chemical quantities. Plant uptake of P and K was generally well correlated with diffusive flux measurements as well as traditional chemical measurements.Contribution from the Western Region, Agricultural Research Service, U.S. Department of Agriculture; and the Montana Agricultural Experiment Station, Bozeman, Montana, submitted as Station Paper No. 654, Journal Series.Soil Scientist, Snake River Conservation Research Center, Kimberly, Idaho; Professor of Chemistry, and Professor of Soils, Montana State University, Bozeman, respectively.     

Calcium flux across disk membranes. Studies with intact rod photoreceptors and purified disks

Calcium accumulation by rod disks was studied in excised bullfrog retinas with 45Ca tracer-exchange methods. Ca uptake by disks is a necessary requirement if light-induced Ca releases from disks mediate photoreceptor excitation. In an hour-long incubation, disks exchanged less than or equal to 0.01 mole of Ca per mole of rhodopsin, or less than or equal to 10% of their total Ca. This corresponds to a unidirectional flux of less than or equal to 0.01 fmol/cm2 S, or less than or equal to 5 ions/disk-second across the disk membrane. Neither incubation in 10 mM Ca (which increases cytoplasmic activity 10--100- fold) nor photostimulation (which photoactivated up to 50% rhodopsin/h) had measurable effect on exchange rate, though an increase of several orders of magnitude would have been expected according to the hypothesis. The observed exchange could not be explained by: (a) 45Ca losses from disks before measurement (neither the net efflux nor the Ca- Ca exchange property of disks adequately explains such losses), (b) a limited pool of exchangeables Ca from strongly binding intradiskal sites, or (c) rate-limiting flux across the plasma membrane during incubation. For the study of the Ca efflux properties of disks, separate experiments were performed with 45Ca-loaded disks. Intradiskal activity could be estimated from the disks' hyperosmotically sensitive 45Ca pool and from their intradiskal volume (indirectly assayed by density). Ca-Ca exchange was undetectable (less than or equal to 0.1 fmol/cm2 S) in disks whose intradiskal activity was at least 0.3 mM. Net efflux was 0.2 fmol/cm2 S for an intradiskal activity of approximately 1 mM and is comparable to published fluxes for phospholipid vesicles. These results seem to exclude the internal space of disks as the source of Ca for photoreceptor excitation.     

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin.

In order to establish whether or not chloride ions behave as freely moving particles in "passive", i.e. ouabain- and acetazolamide-treated, frog skin, tracer fluxes of 36Cl-have been measured while a voltage (generally +40 mV, serosal side positive) across the skin was applied. Ussing's flux ratio equation has been used as a criterion for this type of transport. One group of skin samples exhibited significant exchange diffusion phenomena. Most samples in a second group either behaved according to the flux ratio equation of showed significant and extreme exchange diffusion. From flux ratios obtained at two different voltages across various skin samples, showing extreme exchange diffusion, it appeared that the simple form of Kedem and Essig's law derived from irreversible thermodynamics, which is valid for homogeneous systems, does not apply to the type of exchange diffusion found. The system can, however, be described by a 1:1 exchange mechanism working in parallel with a diffusional pathway. The ratio exchange flux/observed efflux must then have a constant value (0.83) at the voltages appled, which implies that the exchange flux is voltage dependent. By comparison with iodide flux experiments as carried out by Ussing, it is shown that iodide exhibits the same type of exchange diffusion. A carrier, possibly responsible for the observed behaviour, is described.     

Na+-Ca2+ exchange and calcium permeability in canine basolateral membrane vesicles: the effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3',5'-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na+-Ca2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 +/- 1.8% to 20.5 +/- 4.6%, P less than 0.05, n = 6, but had no effect on either passive Ca2+ efflux or sodium-stimulated Ca2+ efflux from Ca2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La3+) but not by the calcium-channel blocker verapamil. La3+ blocked efflux of Ca2+ from preloaded vesicles when it was placed in the external solution. This La3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca2+ uptake at 250 microM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca2+-Ca2+ exchange by the Na+-Ca2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na+-Ca2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.     

Improved 3D continuum calculations of ion flux through membrane channels

A continuum model, based on the Poisson–Nernst–Planck (PNP) theory, is applied to simulate steady-state ion flux through protein channels. The PNP equations are modified to explicitly account (1) for the desolvation of mobile ions in the membrane pore and (2) for effects related to ion sizes. The proposed algorithm for a three-dimensional self-consistent solution of PNP equations, in which final results are refined by a focusing technique, is shown to be suitable for arbitrary channel geometry and arbitrary protein charge distribution. The role of the pore shape and protein charge distribution in formation of basic electrodiffusion properties, such as channel conductivity and selectivity, as well as concentration distributions of mobile ions in the pore region, are illustrated by simulations on model channels. The influence of the ionic strength in the bulk solution and of the externally applied electric field on channel properties are also discussed.     

Spectrophotometric detection of monovalent cation flux in cells: fluorescence microscope measurement of acetylcholine receptor-mediated ion flux in PC-12 cells

A new and convenient spectroscopic method for measuring monovalent cation flux in cells is described. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-dicarboxylic acid) by Cs+. A conventional fluorescence microscope can be used to measure the Cs+ flux. The usefulness of the technique is illustrated by measurement of acetylcholine receptor-mediated Cs+ flux in PC-12 cells, a sympathetic neuronal cell line. The results are the same as those obtained when radioactive tracer ions were used. The technique is applicable to any transmembrane process in which Cs+ can substitute for either Na+ or K+. The method has been developed to identify the different neurotransmitter receptors that control the translocation of monovalent cations and to locate the cells in central nervous system cell preparations that contain these receptors. The advantage of an optical method over tracer ion methods for biochemical and pharmacological studies of transmembrane processes in cells is described.     

Substrate and inhibitor binding and translocation by the platelet plasma membrane serotonin transporter

The Na+ and Cl- dependence of imipramine binding and dissociation were determined in platelet plasma membrane vesicles. Equilibrium imipramine binding affinity depends on Na+ binding to two non-interacting, low-affinity sites. Binding of a single Cl- ion also enhances imipramine affinity. Imipramine dissociation is inhibited by Na+ and Cl-, indicating that both ions can bind after imipramine. Of the two Na+ ions required for imipramine binding, only one is involved in slowing imipramine dissociation, indicating that imipramine binding makes the two Na+ ions non-equivalent. The initial rate of imipramine association is strongly Na(+)-dependent, suggesting that Na+ binds prior to imipramine. Cl-, however, affects imipramine dissociation but not association. Thus, while Na+ and Cl- can bind either before or after imipramine, kinetic considerations impose a most likely binding order of first Na+, then imipramine and finally Cl-. We have confirmed and extended these conclusions using serotonin exchange and efflux measurements. Efflux of radioactivity from vesicles preloaded with [3H]serotonin is stimulated by both external K+ and external unlabelled serotonin. K+ acts to accelerate a step that is rate-limiting for net efflux but that does not involve Na+, Cl- or serotonin translocation. Unlabelled serotonin accelerates radioactivity efflux by exchanging with intravesicular label. This serotonin exchange requires external Cl-, but not external Na+. These results suggest that first Na+, then serotonin and finally Cl- bind from the external medium. Although serotonin exchange requires external Cl-, internal Cl- is not required. These results suggest that translocation does not disturb the spatial order of bound substrates, which dissociate internally in a first-in-first-out order.     

Phosphorimaging detection and quantitation for isotopic ion flux assays

A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated (22)Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated (86)Rb efflux produced data similar to data obtained with the (22)Na influx assay. However, assays for (45)Ca influx were not successful, although (45)Ca was readily detected and quantified. Voltage-gated sodium channel-mediated (22)Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1,200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.     

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)