A Lipid-dependent Uncoupled Conformation of the Acetylcholine Receptor

Lipids influence the ability of Cys-loop receptors to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. With the nicotinic acetylcholine receptor (nAChR) from Torpedo, current models suggest that lipids modulate the natural equilibrium between resting and desensitized conformations. We show that the lipid-inactivated nAChR is not desensitized, instead it adopts a novel conformation where the allosteric coupling between its neurotransmitter-binding sites and transmembrane pore is lost. The uncoupling is accompanied by an unmasking of previously buried residues, suggesting weakened association between structurally intact agonist-binding and transmembrane domains. These data combined with the extensive literature on Cys-loop receptor-lipid interactions suggest that the M4 transmembrane helix plays a key role as a lipid-sensor, translating bilayer properties into altered nAChR function.     

Ligand binding to the membrane-bound acetylcholine receptor from Torpedo marmorata: a complete mathematical analysis.

We have studied by means of equilibrium binding and kinetic experiments the interaction of the membrane-bound nicotinic acetylcholine receptor (nACHR) from Torpedo marmorata with [3H]acetylcholine and the fluorescent agonist NBD-5-acylcholine. In agreement with previous studies by others, we observed the preexistence, in the absence of ligand, of an equilibrium between two states of the nAChR, one with high affinity and the other with low affinity for agonist. As additional requirements for a minimal reaction scheme, we recognized (i) the existence of two ligand-binding sites, each of which may exist in two conformational states when occupied, and (ii) ligand-induced transitions between these conformations. Employing a special form of the allosteric model which considers these requirements, we then developed a suitable algorithm in order to simultaneously fit the whole set of equilibrium binding and kinetic data obtained for the two ligands. In this way we determined for a minimal model of the mechanism of action of the nAChR the complete set of rate constants and KD values involved. With these values available, we were able to simulate the rise and fall in the concentrations of individual receptor-ligand complexes and conformations occurring in the course of excitatory events at the electrocyte synapse. The membrane environment of the nAChR plays a decisive role with respect to the rates of conformational change of the nAChR occurring in the course of ligand interaction. Thus, artificial changes in membrane structure and composition can speed up by several orders of magnitude the rate of conformational change ("desensitization"). A proper structure of the surrounding membrane hence is a prerequisite for the physiological function of the membrane-embedded nAChR.     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Galanthamine and non-competitive inhibitor binding to ACh-binding protein: evidence for a binding site on non-alpha-subunit interfaces of heteromeric neuronal nicotinic receptors

Rapid neurotransmission is mediated through a superfamily of Cys-loop receptors that includes the nicotinic acetylcholine (nAChR), gamma-aminobutyric acid (GABA(A)), serotonin (5-HT(3)) and glycine receptors. A class of ligands, including galanthamine, local anesthetics and certain toxins, interact with nAChRs non-competitively. Suggested modes of action include blockade of the ion channel, modulation from undefined extracellular sites, stabilization of desensitized states, and association with annular or boundary lipid. Alignment of mammalian Cys-loop receptors shows aromatic residues, found in the acetylcholine or ligand-binding pocket of nAChRs, are conserved in all subunit interfaces of neuronal nAChRs, including those that are not formed by alpha subunits on the principal side of the transmitter binding site. The amino-terminal domain containing the ligand recognition site is homologous to the soluble acetylcholine-binding protein (AChBP) from mollusks, an established structural and functional surrogate. We assess ligand specificity and employ X-ray crystallography with AChBP to demonstrate ligand interactions at subunit interfaces lacking vicinal cysteines (i.e. the non-alpha subunit interfaces in nAChRs). Non-competitive nicotinic ligands bind AChBP with high affinity (K(d) 0.015-6 microM). We mutated the vicinal cysteine residues in loop C of AChBP to mimic the non-alpha subunit interfaces of neuronal nAChRs and other Cys loop receptors. Classical nicotinic agonists show a 10-40-fold reduction in binding affinity, whereas binding of ligands known to be non-competitive are not affected. X-ray structures of cocaine and galanthamine bound to AChBP (1.8 A and 2.9 A resolution, respectively) reveal interactions deep within the subunit interface and the absence of a contact surface with the tip of loop C. Hence, in addition to channel blocking, non-competitive interactions with heteromeric neuronal nAChR appear to occur at the non-alpha subunit interface, a site presumed to be similar to that of modulating benzodiazepines on GABA(A) receptors.     

The Nicotinic Acetylcholine Receptor and the Na,K-ATPase α2 Isoform Interact to Regulate Membrane Electrogenesis in Skeletal Muscle

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756–765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639–641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase α2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase α2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase α subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.     

Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid–nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid–nAChR interactions, and highlight potential biological roles for lipid–nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid–protein interactions.     

A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.     

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).     

Incorporation of the nicotinic acetylcholine receptor into planar multilamellar films: characterization by fluorescence and Fourier transform infrared difference spectroscopy.

A method for preparing thin, planar films of nicotinic acetylcholine receptor (nAChR) membranes that retain the ability to undergo the resting to desensitized state transition and that are suitable for spectroscopic studies has been developed. Native, alkaline-extracted nAChR membranes from Torpedo are dried under nitrogen on either a plastic microscope coverslip or a germanium internal reflection element (IRE) and then equilibrated with buffer. The drying procedure has no effect on the functional state of the nAChR as judged by a fluorescence assay using the probe ethidium bromide. The times required for an acetylcholine analogue (carbamylcholine), a local anesthetic (dibucaine), and a fluorescent probe (ethidium bromide) to penetrate films of varying degrees of thickness, interact with the receptor, and then to be washed from the films have been established. Under these conditions, the nAChR films can be repetitively cycled between the resting and desensitized states. Both fluorescence and infrared spectroscopy show that the films adhere strongly to either support even with buffer flowing continuously past the film surface. Fourier transform infrared difference spectra calculated from spectra recorded in the presence and absence of carbamylcholine show small, reproducible bands which reflect changes in nAChR structure upon desensitization.     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Synthesis and biological evaluation of novel carbon-11 labeled pyridyl ethers: candidate ligands for in vivo imaging of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in the brain with positron emission tomography

The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope ¹¹C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics.     

Synthesis and evaluation of a conditionally-silent agonist for the α7 nicotinic acetylcholine receptor

We introduce the term ‘silent agonists’ to describe ligands that can place the α7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5′-phenylanabaseine) was synthesized and identified as a new silent agonist for the α7 nAChR; it binds to the receptor but does not activate α7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C–C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the α7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of α7 nAChR ligands may constitute a new alternative for the development of α7 nAChR therapeutics.     

Molecular mechanisms of acetylcholine receptor–lipid interactions: from model membranes to human biology

Lipids are potent modulators of the Torpedo nicotinic acetylcholine receptor. Lipids influence nicotinic receptor function by allosteric mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations. Recent structures reveal that lipids could alter function by modulating transmembrane α-helix/α-helix packing, which in turn could alter the conformation of the allosteric interface that links the agonist-binding and transmembrane pore domains—this interface is essential in the coupling of agonist binding to channel gating. We discuss potential mechanisms by which lipids stabilize different conformational states in the context of the hypothesis that lipid–nicotinic receptor interactions modulate receptor function at biological synapses.     

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.     

Dissection of N,N-diethyl-N′-phenylpiperazines as α7 nicotinic receptor silent agonists

The α7 nicotinic acetylcholine receptor (nAChR) is a target for control of inflammation-related phenomena via compounds that are able to selectively induce desensitized states of the receptor. Compounds that selectively desensitize, without facilitating significant channel activation, are termed ‘silent agonists’ because they can be discriminated from antagonists by the currents evoked with co-application with type II positive allosteric modulators (PAMs). One example is N,N-diethyl-N′-phenyl-piperazine (diEPP) (J. Pharm. Exp. Ther. 2014, 350, 665). We used Ullmann-type aryl amination to synthesize a panel of 27 compounds related to diEPP by substitutions at the aryl ring and in the linkage between the piperazine and phenyl rings. Two-electrode voltage clamping of the human α7 nAChR expressed in Xenopus oocytes revealed that it was possible to tune the behavior of compounds to show enhanced desensitization without corresponding partial agonist activity such that trifluoromethyl and carboxamide aryl substituents showed 33 to 46-fold larger PAM-dependent net-charge responses, indicating selective partitioning of the ligand receptor complexes into the desensitized state.     

Mechanism-based approach to the successful prevention of cocaine inhibition of the neuronal (alpha 3 beta 4) nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a family of five channel-forming proteins that regulate communication between the approximately 10(12) cells of the nervous system. A minimum mechanism of inhibition of the muscle-type nAChR (1) by the noncompetitive inhibitors cocaine and MK-801 [(+)-dizocilpine, an anticonvulsant] indicated they bind to a regulatory site, with higher affinity for the closed-channel form than for the open-channel form, thus shifting the equilibrium toward the closed-channel form and inhibiting receptor function. The mechanism predicts that compounds that bind to this regulatory site with equal or higher affinity for the open-channel conformation than for the closed-channel conformation will prevent receptor inhibition (1). Does a neuronal form of the receptor behave similarly? The mechanism of inhibition of the neuronal nAChR by cocaine and MK-801 using rapid chemical kinetic techniques was investigated. The alpha3beta4 nAChR stably expressed in HEK 293 cells was used in these investigations. Whole-cell currents originated from a major and minor nAChR isoform. Only the major isoform has been characterized. For the dominant, rapidly desensitizing isoform, the carbamoylcholine dissociation constant for the site controlling receptor activation, Kd, is 2 mM; the channel-opening equilibrium constant, Phi(-1), is 4; and the dominant desensitization rate constant, k34, is 20 s(-1). Cocaine inhibits the receptor noncompetitively, with an apparent KI of 84 and 26 microM at high and low carbamoylcholine concentrations, at which concentrations the receptor is mainly in the open- or closed-channel form, respectively. Similar results were obtained with MK-801. A combinatorially synthesized RNA ligand and a cocaine analogue alleviated cocaine inhibition of this neuronal receptor.     

Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein

The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.     

Equilibrium binding of cholinergic ligands to the membrane-bound acetylcholine receptor

We have studied the binding equilibria of the membrane-bound acetylcholine receptor from Torpedo marmorata with representative cholinergic ligands by means of two fluorescence and a rapid centrifugation assay. Based on the established mechanism of acetylcholine binding to the receptor (Fels, G., Wolff, E. K., and Maelicke, A. (1982) Eur. J. Biochem. 127, 31-38), the obtained binding and competition data were analyzed assuming two classes of interacting sites for all ligands studied. The experimental data were consistent with this assumption and, based on the obtained KD values, suggest weak positively cooperative interactions of binding sites when occupied by agonists but independent (or negatively cooperative interacting) sites when occupied by antagonists. Based on the fluorescence binding assay employed, agonists and antagonists induce different conformational states of the liganded receptor. These states seem to be similar for all antagonists tested but differ for the different agonists tested. The existence of ligand-specific conformational states suggests a close link of these states with receptor function.     

Electrostatic interactions regulate desensitization of the nicotinic acetylcholine receptor

To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.