Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor

Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.     

Channel permeant cations compete selectively with noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Previous work suggests that noncompetitive inhibitor (NCI) ligands and channel permeant cations bind to sites within the nicotinic acetylcholine receptor ion channel. We have used ethidium as a fluorescent probe of the NCI site to investigate interactions between NCI ligands and channel permeant cations. We found that ethidium can be completely displaced from the receptor by a variety of inorganic monovalent and divalent cations. The rank order of monovalent cation affinities was found to be Tl+ greater than Rb+ greater than or equal to K+ greater than Cs+ greater than Na+ greater than Li+. The monovalent cation Kd values vary markedly over a 40-fold range, from 3 to 121 mM. The Kd values and rank order correspond to values determined previously from electrophysiological data. Hill plots of the back titrations yield slopes of 1.0 for all monovalent cations, indicating a single class of independent sites, as shown previously for NCI ligands. Scatchard analysis of ethidium binding in the presence of Tl+ reveals a reduction in affinity and no changes in the maximal number of sites. In the presence of agonist the kinetics of ethidium dissociation induced by the addition of phencyclidine or cations alone or the simultaneous addition of both are nearly identical. The ethidium dissociation rate induced by either phencyclidine or cations is regulated by the occupation of the agonist sites in a similar manner. These results indicate that the effect of cations on NCI ligand binding occurs by mutually exclusive competition. We suggest that NCIs can regulate cation binding at a physiological cation recognition site that is likely part of the cation permeation path through the receptor channel.     

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.     

Location of the polyamine binding site in the vestibule of the nicotinic acetylcholine receptor ion channel

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.     

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.     

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.     

N-methylcytisine--a selective ligand of nicotinic receptors of acetylcholine in the CNS

The ability of cytisine and its N-methyl derivatives to bind to nicotinic acetylcholine receptors (nAChR) from different tissues was studied. Cytisine and N-methylcytisine have high affinity (KD = 50 nM) to nAChR from squid optical ganglia. N,N-dimethylcytisine did not show high affinity to this receptor. In the case of nAChR from T. marmorata, cytisine was the only effective inhibitor of 14C-tubocurarine specific binding (Ki = 700 nM). N-methyl- and N,N-dimethylcytisine did not displace 14C-tubocurarine at a concentration of 0.1 mM. The results obtained indicate that there are some differences in the structure of nAChR binding sites from squid and T. marmorata optical ganglia.     

The n-alcohol site in the nicotinic receptor pore is a hydrophobic patch

Alcohols and volatile anesthetics inhibit peripheral nicotinic acetylcholine receptors noncompetitively, primarily via an open-channel block mechanism. Analysis of hydrophobic mutations near the middle of the pore-forming M2 domains suggested that alcohols interact with the pore in this vicinity. To establish the extent of this inhibitory site, we created a series of hydrophobicity-altering mutations scanning most of the alpha subunit M2 domain. Using both single-channel and rapid patch perfusion electrophysiology, we measured how these mutations affect nAChR sensitivity to ethanol and hexanol. We find a near-contiguous series of amino acids in alpha-M2, extending from alphaL250 (8') to alphaV255 (13'), where mutagenesis strongly influences inhibition by alcohols. These results support the existence of a large inhibitory patch in the nAChR pore lining where interactions with alcohols are primarily due to hydrophobic forces. Ethanol appears to interact with deeper regions of this site than does hexanol. Because alcohols apparently act as open-channel blockers, we infer from our results that most of the residues between alphaL250 and alphaV255 are exposed to the aqueous environment of the pore when the channel is open. The location and extent of this site can explain why small alcohols occupy the nAChR pore at the same time as larger alcohols or charged blockers, while two large alcohols bind in a mutually exclusive manner.     

Ligand-activated ion channels may share common gating mechanisms with the Shaker potassium channel.

This paper proposes a detailed gating mechanism for the N-methyl-D-aspartate (NMDA) channel. In the NMDAR1 subunit, the signal of agonist binding may be carried from Y456 to W590 through an electron transport chain, including W480 which could be the glycine modulatory site. The channel's opening may arise from repulsion between negatively charged W590s, analogous to W435s of the Shaker K+ channel. The cyclic nucleotide-gated channels may be activated by a similar mechanism, but the opening of nicotinic acetylcholine receptor (nAChR) channels is likely to be initiated by the formation of tyrosine radicals. The role of disulfide-bonded cysteines in the redox modulation can also be explained.     

A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the 'Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, gamma-aminobutyric acid and serotonin. Cryo-electron microscopy has yielded a three-dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 A. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height about 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 A radius hydrophobic pore can form a functional barrier to the permeation of a 1 A radius Na+ ion. Using a united-atom force field for the protein instead of an all-atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.     

Synthetic polyamines: an overview of their multiple biological activities

The binding of polyamines to a variety of receptors and other defined recognition sites has been widely reported. It is well-known that polyamines interact with aspartate, glutamate, and aromatic residues of a given receptor and/or enzyme mainly through the formation of ion bonds, since at physiological pH, protonation of amino groups is nearly complete. From this, the hypothesis arises that a polyamine may be a universal template able to recognize different receptor systems. This hypothesis suggests that both affinity and selectivity may be fine-tuned by inserting appropriate substituents onto the amine functions and by varying the methylene chain lengths between them on the polyamine backbone. In this paper, we detail several application of this design strategy aimed at discovering potent and selective polyamines able to bind neurotransmitter receptors and enzymes, such as muscarinic receptor subtypes, muscle-type nicotinic receptors and acethylcholinesterase.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.     

Identification of Purine Binding Sites on Torpedo Acetylcholine Receptor

Abstract

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[³H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the β-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.     

Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.     

Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis

Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.     

Molecular Docking and Dynamic Simulation to Identify α7nAChR Binding Affinity of Flavonoids for the Treatment of Alzheimer's Disease

Background : Alpha-7-nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel is one of the important parts of the cholinergic pathway in the brain and has a remarkable role in Alzheimer's disease (AD). It has been documented that the modulation of α7nAChR with the help of phytoconstituent can be helpful in the treatment of AD. Method : The binding efficacy of fifty flavonoids was evaluated for human α7nAChR using molecular docking. The best two flavonoids shortlisted from docking analysis were then subjected to molecular dynamic simulations for 100 ns to analyze conformational binding stability with the target protein. Further, the druggability of the selected flavonoids was checked using in silico ADMET studies. Result : The top two flavonoids selected based on binding affinity toward the binding site of α7nAChR from molecular docking were amentoflavone (–9.1 kcal/mol) and gallocatechin (–8.8 kcal/mol). The molecular dynamics simulation revealed that amentoflavone and gallocatechin have a stable state during overall simulation time, lesser root mean deviation (RMSD) and root mean square fluctuation (RMSF), and complex of both compounds with protein is stable until 100 ns. Conclusion : The two flavonoids amentoflavone and gallocatechin are potential lead molecules that could be utilized as effective agonists of α7nAChR to combat Alzheimer's disease. Future in vitro and in vivo analyses are required to confirm their effectiveness.     

Preparation and biological properties of biotinylated PhTX derivatives.

We report the synthesis of several highly functionalized biotinylated philanthotoxin (PhTX) analogues (7, 8, 10, 13-16) designed on the basis of earlier structure-activity relationship studies. Despite the extensive modifications, the binding to nicotinic acetylcholine receptor (nAChR) is in the low micromolar range according to an inhibition assay using 3H-thienylcyclohexyl-piperidine (TCP). A patch clamp functional assay gave comparable results. Compounds exemplified by 16, which consists of a biotinylated ligand linked to a bifunctional photoaffinity probe (BPP), represent a new type of probe which should find use in photo-crosslinking studies of ligand receptor interactions.     

Synthesis and evaluation of xanomeline analogs--probing the wash-resistant phenomenon at the M1 muscarinic acetylcholine receptor

A series of xanomeline analogs were synthesized and evaluated for binding at the M(1) muscarinic acetylcholine receptor (M(1) receptor). Specifically, compounds that substitute the O-hexyl chain of xanomeline with polar, ionizable, or conformationally restricted moieties were assessed for their ability to bind to the M(1) receptor in a wash-resistant manner (persistent binding). From our screen, several novel ligands that persistently bind to the M(1) receptor with greater affinity than xanomeline were discovered. Results indicate that persistent binding may arise not only from hydrophobic interactions but also from ionic interactions with a secondary M(1) receptor binding site. Herein, a qualitative model that accounts for both binding scenarios is proposed and applied to understand the structural basis to wash-resistant binding and long-acting effects of xanomeline-based compounds.