Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are pentameric proteins that belong to the Cys-loop receptor superfamily. Their essential mechanism of functioning is to couple neurotransmitter binding, which occurs at the extracellular domain, to the opening of the membrane-spanning cation channel. The function of these receptors can be modulated by structurally different compounds called noncompetitive antagonists. Noncompetitive antagonists may act at least by two different mechanisms: a steric and/or an allosteric mechanism. The simplest idea representing a steric mechanism is that the antagonist molecule physically blocks the ion channel. On the other hand, there exist distinct allosteric mechanisms. For example, noncompetitive antagonists may bind to the receptor and stabilize a nonconducting conformational state (e.g., resting or desensitized state), and/or increase the receptor desensitization rate. Barbiturates, dissociative anesthetics, antidepressants, and neurosteroids have been shown to inhibit nicotinic receptors by allosteric mechanisms and/or by open- and closed-channel blockade. Receptor modulation has proved to be highly complex for most noncompetitive antagonists. Noncompetitive antagonists may act by more than one mechanism and at distinct sites in the same receptor subtype. The binding site location for one particular molecule depends on the conformational state of the receptor. The mechanisms of action and binding affinities of noncompetitive antagonists differ among nicotinic receptor subtypes. Knowledge of the structure of the nicotinic acetylcholine receptor, the location of its noncompetitive antagonist binding sites, and the mechanisms of inhibition will aid the design of new and more efficacious drugs for treatment of neurological diseases.     

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.     

Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor

Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded KD values of 2-4 microM for the desensitized AChR and approximately 600 microM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Examining the noncompetitive antagonist-binding site in the ion channel of the nicotinic acetylcholine receptor in the resting state

3-Trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) has been shown to be a potent noncompetitive antagonist (NCA) of the nicotinic acetylcholine receptor (AChR). Amino acids that contribute to the binding site for [(125)I]TID in the ion channel have been identified in both the resting and desensitized state of the AChR (White, B.H., and Cohen, J.B. (1992) J. Biol. Chem. 267, 15770-15783). To characterize further the structure of the NCA-binding site in the resting state channel, we have employed structural analogs of TID. The TID analogs were assessed by the following: 1) their ability to inhibit [(125)I]TID photoincorporation into the resting state channel; 2) the pattern, agonist sensitivity, and NCA inhibition of [(125)I]TID analog photoincorporation into AChR subunits. The addition of a primary alcohol group to TID has no demonstrable effect on the interaction of the compound with the resting state channel. However, conversion of the alcohol function to acetate, isobutyl acetate (TIDBIBA), or to trimethyl acetate leads to rightward shifts in the concentration-response curves for inhibition of [(125)I]TID photoincorporation into the AChR channel and a progressive reduction in the agonist sensitivity of [(125)I]TID analog photoincorporation into AChR subunits. Inhibition of [(125)I]TID analog photoincorporation by NCAs (e.g. tetracaine) as well as identification of the sites of [(125)I]TIDBIBA photoincorporation in the deltaM2 segment indicate a common binding locus for each TID analog. We conclude that relatively small additions to TID progressively reduce its ability to interact with the NCA site in the resting state channel. A model of the NCA site and resting state channel is presented.     

5-Doxylstearate-induced displacement of phencyclidine from its low-affinity binding sites on the nicotinic acetylcholine receptor.

Fatty acids as well as phencyclidine (PCP) inhibit the ion channel activity of the nicotinic acetylcholine receptor (AChR) by a noncompetitive mechanism. However, the exact localization of the fatty acid binding sites is unknown and, thus, the noncompetitive inhibitory mechanism for these endogenous modulators remains to be elucidated. In an attempt to determine the location of the fatty acid binding sites, we study the mutually exclusive action between 5-doxylstearate (5-SASL), a derivative of the endogenous noncompetitive antagonist (NCA) stearic acid, and other exogenous NCAs. For this purpose, both equilibrium and competitive binding assays using fluorescent and radiolabeled ligands were performed on desensitized AChRs. More specifically, we determined: (i) the effect of 5-SASL on the binding of the exogenous NCA [(3)H]PCP; (ii) the effect of 5-SASL on the binding of either quinacrine or ethidium, two fluorescent NCAs from exogenous origin; and (iii) the PCP-induced displacement of quinacrine and ethidium from their respective high-affinity binding sites. Our first target (i) is carried out by measuring the [(3)H]PCP binding in the absence or in the presence of increasing concentrations of 5-SASL. We found that 5-SASL displaces PCP from its low-affinity binding sites. The low-affinity PCP binding sites were pharmacologically characterized by an apparent dissociation constant (K(d)) of 6.1 +/- 5.0 microM and a stoichiometry of 3.7 +/- 1.5 sites per AChR. The fact that 5-SASL increased the apparent K(d) without changing the number of sites per AChR is indicative of a mutually exclusive action. From these results, an apparent inhibition constant (K(i)) of 75 +/- 31 microM for 5-SASL was calculated. In addition, 5-SASL affected neither the apparent K(d) (0.46 +/- 0.37 microM) nor the stoichiometry (1.07 +/- 0.57 sites per AChR) of the high-affinity PCP binding site. The second objective (ii) is achieved by titrating either quinacrine or ethidium into AChR native membranes in the absence or in the presence of increasing concentrations of 5-SASL. These experiments showed that 5-SASL efficiently increased the apparent K(d) of quinacrine without perturbing the interaction of ethidium with its high-affinity locus. Considering that (a) 5-SASL effectively quenched the AChR-bound quinacrine fluorescence (H. R. Arias, Biochim. Biophys. Acta 1347, 9-22, 1997) and (b) fluorescence-quenching is a short-range process, it is possible to suggest that 5-SASL displaces quinacrine from its high-affinity binding site by a steric mechanism. In this regard, a K(i) of 38 +/- 5 microM for 5-SASL was calculated. Concerning the last objective (iii), AChR-bound quinacrine or ethidium was back titrated with PCP. Two PCP K(i) values were obtained by fitting the displacement plots by nonlinear regression with two components. The lowest K(i) values obtained for either quinacrine (0.86 +/- 0.37 microM) or ethidium (0. 29 +/- 0.23 microM) displacement from their respective high-affinity binding sites coincide with the previously determined high-affinity [(3)H]PCP K(d). In addition, the highest K(i) values obtained for either NCA displacement are in the same concentration range as the observed low-affinity [(3)H]PCP K(d). Taking into account all experimental data, we reached the following conclusions: (i) fatty acid molecules, or at least 5-SASL, sterically interact with both the PCP low-affinity and the quinacrine high-affinity binding sites; (ii) the low-affinity PCP binding sites, as well as the high-affinity quinacrine locus, are located at the nonannular lipid domain of the AChR; and, finally, (iii) fatty acid molecules are not accessible to the lumen of the ion channel, indicating an allosteric mode of action for fatty acids to inhibit ion flux. Thus, the 5-SASL, the quinacrine high-affinity, and the PCP low-affinity binding sites are all located at overlapping nonannular loci on the muscle-type AChR.     

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.     

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca²⁺ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [³H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6′) and valine (position 13′) rings, and (c) inhibits [³H]TCP, [³H]ibogaine, and [³H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.     

Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca²⁺ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca²⁺ influx in hα3β4 AChRs with ～9-fold higher potency than that for PCP, (b) [³H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (K_d = 0.46 ± 0.06 μM), and ibogaine inhibits [³H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [³H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.     

Identifying the binding site(s) for antidepressants on the Torpedo nicotinic acetylcholine receptor: [3H]2-azidoimipramine photolabeling and molecular dynamics studies

Radioligand binding, photoaffinity labeling, and docking and molecular dynamics were used to characterize the tricyclic antidepressant (TCA) binding sites in the nicotinic acetylcholine receptor (nAChR). Competition experiments indicate that the noncompetitive antagonist phencyclidine (PCP) inhibits [3H]imipramine binding to resting (closed) and desensitized nAChRs. [3H]2-azidoimipramine photoincorporates into each subunit from the desensitized nAChR with approximately 25% of the labeling specifically inhibited by TCP (a PCP analog), whereas no TCP-inhibitable labeling was observed in the resting (closed) state. For the desensitized nAChR and within the alpha subunit, the majority of specific [3H]2-azidoimipramine labeling mapped to a approximately 20 kDa Staphylococcus aureus V8 protease fragment (alphaV8-20; Ser173-Glu338). To further map the labeling site, the alphaV8-20 fragment was further digested with endoproteinase Lys-C and resolved by Tricine SDS-PAGE. The principal labeled fragment (11 kDa) was further purified by rpHPLC and subjected to N-terminal sequencing. Based on the amino terminus (alphaMet243) and apparent molecular weight, the 11 kDa fragment contains the channel lining M2 segment. Finally, docking and molecular dynamics results indicate that imipramine and PCP interact preferably with the M2 transmembrane segments in the middle of the ion channel. Collectively, these results are consistent with a model where PCP and TCA bind to overlapping sites within the lumen of the Torpedo nAChR ion channel.     

Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch-clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.     

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.     

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.     

(−)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites

The interaction of (−)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (−)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca²⁺ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC₅₀ = 3.86 ± 0.49 and 1.92 ± 0.48 μM, respectively, (b) binds to the [³H]TCP site with ∼13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [³H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6′ and 13′ in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9′ is the minimum structural component for (−)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (−)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants.     

Modulation of nicotinic acetylcholine receptor conformational state by free fatty acids and steroids

Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the K(D) of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in K(D) in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A(2) treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the gammaM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.     

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.     

Structure-activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca2? influx in human embryonic muscle AChRs with the following potency sequence (IC(50) in μM): (±)-18-methylaminocoronaridine (5.9±0.3)～(±)-18-methoxycoronaridine (18-MC) (6.8±0.8)>(-)-ibogaine (17±3)～(+)-catharanthine (20±1)>(±)-albifloranine (46±13), (b) bind to the [3H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [3H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [3H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC??) that correlate very well with the pK(i) values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ～345 ?3 for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization.     

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine ([(125)I]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [(125)I]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [(14)C]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [(3)H]tetracaine binding (K(i)s approximately 20.9 and 2.0 microM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.     

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.     

Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor

The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.