Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.     

Roles of agonist-binding sites in nicotinic acetylcholine receptor function

Under equilibrium conditions, the nicotinic acetylcholine receptor from Torpedo electroplax carries two high affinity-binding sites for agonists. It is generally assumed that these are the only agonist sites on the receptor and that their occupancy results in rapid channel activation followed by slower conformational transitions that lead to the high affinity equilibrium state. These slow transitions are thought to reflect the physiological process of desensitization. Here we show that preequilibration of the high affinity sites with saturating concentrations of carbamylcholine does not diminish the ion flux response to subsequent exposure to higher (activating) concentrations of this agonist. This finding has profound implications with respect to receptor function: (1) occupancy of the high affinity sites per se does not desensitize the receptor and (2) these sites cannot be directly involved in receptor activation. It is thus necessary to invoke the presence of additional binding sites in channel opening.     

Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 m M ). Substance P had no effect on the initial rate of ¹²⁵I-α-bungarotoxin binding at concentrations of <100 μ M . The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [³H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [³H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.     

Agonist-induced affinity alterations of a central nervous system alpha-bungarotoxin receptor

Abstract— The ability of cholinergic agonists to block the specific interaction of α-bungarotoxin (α-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of α-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of α-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.     

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of thiol modification and Ca++ on agonist-specific state transitions of nicotinic acetylcholine receptor from Torpedo californica electroplax.

The agonist binding affinity of nicotinic acetylcholine receptor (nAChR) from $\text{Torpedo}$$\text{californica}$ electroplax, as inferred from ability of agonist to inhibit specific curaremimetic neurotoxin binding to nAChR, is sensitive to the duration of exposure to agonist. The concentration of carbachol necessary to prevent one-half of toxin binding over a 30 min incubation with nAChR (K₃₀) is 10 μM when toxin and carbachol are simultaneously added to membrane-bound nAChR, and 3 μM when nAChR are pretreated with carbachol for 30 min prior to the addition of toxin. These alterations in agonist affinity may be mimicked by modification of nAChR thiol groups. Affinity of nAChR for carbachol is decreased following treatment with dithiothreitol (DTT). Dithio-bis-nitrobenzoic acid treatment of DTT-reduced membranes yields K₃₀ values of 5 μM for carbachol, while N-ethylmaleimide treatment of DTT-reduced nAChR produces nAChR with reduced affinity for carbachol, reflected in K₃₀ values of about 400 μM. In the absence of Ca⁺⁺, K₃₀ values for carbachol binding to native and DTT-reduced nAChR are diminished 3–6 fold. These affinity alterations are not observed with d-tubocurarine (antagonist) binding to nAChR. Thus, Ca⁺⁺ and the oxidation state of nAChR thiols appear to affect the affinity of nAChR for agonists (but not antagonists), and may therefore be related to agonist-mediated events in receptor activation and/or desensitization.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

Desensitization of NMDA receptor channels is modulated by glutamate agonists

Two distinct forms of desensitization have been characterized for N-methyl-D-aspartate (NMDA) receptors. One form results from a weakening of agonist affinity when channels are activated whereas the other form of desensitization results when channels enter a long-lived nonconducting state. A weakening of glycine affinity upon NMDA receptor activation has been reported. Cyclic reaction schemes for NMDA receptor activation require that a concomitant affinity shift should be observed for glutamate agonists. In this study, measurements of peak and steady-state NMDA receptor currents yielded EC50 values for glutamate that differed by 1.9-fold, but no differences were found for another agonist, L-cysteine-S-sulfate (LCSS). Simulations show that shifts in EC50 values may be masked by significant degrees of desensitization resulting from channels entering a long-lived nonconducting state. Simulations also show that a decrease in the degree of desensitization with increasing agonist concentration is a good indicator for the existence of desensitization resulting from a weakening of agonist affinity. Both glutamate and LCSS exhibited this trend. An affinity difference of three- to eightfold between high-and low-affinity agonist-binding states was estimated from fitting of dose-response data with models containing both types of desensitization. This indicates that activation of NMDA receptors causes a reduction in both glutamate and glycine affinities.     

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.     

Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effect of modulators

1. Loss of response after prolonged or repeated application of stimulus is generally termed desensitization. A wide variety of phenomena occurring in living organisms falls under this general definition of desensitization. There are two main types of desensitization processes: specific and non-specific. 2. Desensitization of the nicotinic acetylcholine receptor is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. It is a case of specific desensitization and is an intrinsic molecular property of the receptor. 3. Desensitization of the nicotinic acetylcholine receptor at the neuromuscular junction was first reported by Katz and Thesleff in 1957. Desensitization of the receptor has been demonstrated by rapid kinetic techniques and also by the characteristic "burst kinetics" obtained from single-channel recordings of receptor activity in native as well as in reconstituted membranes. In spite of a number of studies, the detailed molecular mechanism of the nicotinic acetylcholine receptor desensitization is not known with certainty. The progress of desensitization is accompanied by an increase in affinity of the receptor for its agonist. This change in affinity is attributed to a conformational change of the receptor, as detected by spectroscopic and kinetic studies. A four-state general model is consistent with the major experimental observations. 4. Desensitization of the nicotinic acetylcholine receptor can be potentially modulated by exogenous and endogenous substances and by covalent modifications of the receptor structure. Modulators include the noncompetitive blockers, calcium, the thymic hormone peptides (thymopoietin and thymopentin), substance P, the calcitonin gene-related peptide, and receptor phosphorylation. Phosphorylation is an important posttranslational covalent modification that is correlated with the regulation and desensitization of the receptor through various protein kinases. 5. Although the physiological significance of desensitization of the nicotinic receptor is not yet fully understood, desensitization of receptors probably plays a significant role in the operation of the neuronal networks associated in memory and learning processes. Desensitization of the nicotinic receptor could also possibly be related to the neuromuscular disease, myasthenia gravis.     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

Calcium Modulation of Activation and Desensitization of Nicotinic Receptors from Mouse Brain

Abstract: The effects of extracellular calcium on functional properties of nicotinic receptors from mouse thalamus were investigated. Previous studies have reported that calcium modulates the function of several neuronal nicotinic receptors. A ⁸⁶Rb⁺ ion efflux assay was developed to measure nicotinic receptor function from brain tissue, and data indicate that α₄β₂ receptors may mediate this response. Using the ⁸⁶Rb⁺ efflux assay, calcium effects on receptor activation, desensitization induced by high, activating and low, subactivating concentrations of agonist, and recovery from desensitization were examined. Effects of calcium on the kinetics of ligand binding were also investigated. Calcium modulated receptor activation by increasing the maximal response to nicotine in a concentration-dependent manner, without affecting the EC₅₀ of nicotine. Barium, but not magnesium, mimicked the effects of calcium on receptor activation. The increase in receptor activation could not be explained by changes in the ratio of activatable to desensitized receptors as assessed by the kinetics of ligand binding. Desensitization following activation was unaffected by calcium. Calcium, barium, and magnesium, however, increased the potency of nicotine for desensitization induced by exposure to low, subactivating concentrations of nicotine. Recovery from desensitization was not modulated by calcium. These data suggest that calcium modulates various functional aspects of nicotinic receptors from mouse brain and may do so via different mechanisms.     

Ligand-specific state transitions of the membrane-bound acetylcholine receptor

We have developed a simple, direct and time-resolved method to monitor ligand-induced changes in agonist affinity of the membrane-bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD-5-acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD-5-acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half-life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t1/2 approximately 60 s at 22 degrees C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non-competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.     

Modulation of the binding affinity of myelopoietins for the interleukin-3 receptor by the granulocyte colony-stimulating factor receptor agonist.

Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation, and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.     

A binding site tyrosine shapes desensitization kinetics and agonist potency at GluR2. A mutagenic, kinetic, and crystallographic study

Binding of an agonist to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA) receptor family of the glutamate receptors (GluRs) results in rapid activation of an ion channel. Continuous application results in a non-desensitizing response for agonists like kainate, whereas most other agonists, such as the endogenous agonist (S)-glutamate, induce desensitization. We demonstrate that a highly conserved tyrosine, forming a wedge between the agonist and the N-terminal part of the bi-lobed ligand-binding site, plays a key role in the receptor kinetics as well as agonist potency and selectivity. The AMPA receptor GluR2, with mutations in Tyr-450, were expressed in Xenopus laevis oocytes and characterized in a two-electrode voltage clamp setup. The mutation GluR2(Y450A) renders the receptor highly kainate selective, and rapid application of kainate to outside-out patches induced strongly desensitizing currents. When Tyr-450 was substituted with the larger tryptophan, the (S)-glutamate desensitization is attenuated with a 10-fold increase in steady-state/peak currents (19% compared with 1.9% at the wild type). Furthermore, the tryptophan mutant was introduced into the GluR2-S1S2J ligand binding core construct and co-crystallized with kainate, and the 2.1-A x-ray structure revealed a slightly more closed ligand binding core as compared with the wild-type complex. Through genetic manipulations combined with structural and electrophysiological analysis, we report that mutations in position 450 invert the potency of two central agonists while concurrently strongly shaping the agonist efficacy and the desensitization kinetics of the AMPA receptor GluR2.     

7-Azaindole derivatives as potential partial nicotinic agonists

We have investigated a series of 7-azaindoles as potential partial agonists of the alpha4beta2 nicotinic acetylcholine receptor (nAChR). Three series of 7-azaindole derivatives have been synthesized and evaluated for rat brain neuronal nicotinic receptor affinity and functional activity. Compound (+)-51 exhibited the most potent nAChR binding (Ki = 10 nM). Compound 30A demonstrated both moderate binding affinity and partial agonist potency, thus representing a promising lead for the indications of cognition and smoking cessation.     

Low affinity of beta-adrenergic receptors for agonists on intact cells is not due to receptor sequestration

The low affinity of beta-adrenergic receptors for agonists described on intact cells at 37 degrees C has usually been interpreted in terms of reduced accessibility of agonists (which are usually hydrophilic) for sequestered receptors. We challenged this hypothesis by eliminating the plasma membrane barrier with low doses of the detergent digitonin. In human mononuclear leukocytes (MNL) permeabilized with digitonin, sequestered receptors became accessible to hydrophilic ligands such as agonists, but the affinity was still low. Then we investigated the relationship between low affinity agonist binding and sequestration using concanavalin A, which blocks sequestration. Even when sequestration was blocked, the affinity of the beta-adrenergic receptors for agonists was low. We conclude that: (a) low affinity agonist binding is independent of receptor sequestration; (b) the receptors which undergo conformational change are those that are sequestered; (c) the low affinity appears before sequestration occurs. This receptor conformational change could be the first step in agonist-induced desensitization.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.