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.     

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.     

(−)-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.     

Selectivity of coronaridine congeners at nicotinic acetylcholine receptors and inhibitory activity on mouse medial habenula

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca²⁺ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca²⁺ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [³H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities.     

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.     

Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors

Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit hα1β1γδ AChR-induced Ca²⁺ influx with higher potencies than dizocilpine. The potency of fluoxetine is increased ～10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [³H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-³H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [³H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13′) and leucine (position 9′) rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20′, 21′, and 24′. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20′) rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization.     

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone,a photoreactive bupropion derivative,with nicotinic acetylcholine receptors

The pharmacological properties of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13′) and serine (position 6′) rings. However, an additional domain, between the outer (position 20′) and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.     

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone,a photoreactive bupropion derivative,with nicotinic acetylcholine receptors

The pharmacological properties of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13′) and serine (position 6′) rings. However, an additional domain, between the outer (position 20′) and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.     

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.     

Bupropion binds to two sites in the Torpedo nicotinic acetylcholine receptor transmembrane domain: a photoaffinity labeling study with the bupropion analogue [(125)I]-SADU-3-72

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(N-tert-butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone (SADU-3-72). Based on inhibition of [(125)I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC(50) = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC(50) = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [(125)I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [(125)I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu(265), βLeu(257)) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr(213) in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.     

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.     

Molecular mechanisms and binding site location for the noncompetitive antagonist crystal violet on nicotinic acetylcholine receptors

We investigated the molecular mechanisms and the binding site location for the fluorophor crystal violet (CrV), a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR). To this end, radiolabeled competition binding, fluorescence spectroscopy, Schild-type analysis, patch-clamp recordings, and molecular dynamics approaches were used. The results indicate that (i) CrV interacts with the desensitized Torpedo AChR with higher affinity than with the resting state at several temperatures (5-37 degrees C); (ii) CrV-induced inhibition of the phencyclidine (PCP) analogue [(3)H]thienylcyclohexylpiperidine binding to the desensitized or resting AChR is mediated by a steric mechanism; (iii) tetracaine inhibits CrV binding to the resting AChR, probably by a steric mechanism; (iv) barbiturates modulate CrV binding to the resting AChR by an allosteric mechanism; (v) CrV itself induces AChR desensitization; (vi) CrV decreases the peak of macroscopic currents by acting on the resting AChR but without affecting the desensitization rate from the open state; and (vii) two tertiary amino groups from CrV may bind to the alpha1-Glu(262) residues (located at position 20') in the resting state. We conclude that the CrV binding site overlaps the PCP locus in the resting and desensitized state. The noncompetitive action of CrV may be explained by an allosteric mechanism in which the binding of CrV to the extracellular mouth of the resting receptor leads to an inhibition of channel opening. Binding of CrV probably increases desensitization of the resting channel and stabilizes the desensitized state.     

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.     

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.     

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.     

High affinity ibogaine binding to a mu opioid agonist site

The naturally occurring indole alkaloid ibogaine is of interest because of its reported ability to block drug seeking behavior for extended periods. The compound also potentiates morphine-induced analgesia in mice and reduces certain naltrexone-precipitated withdrawal signs in morphine-dependent rats. Although these results might suggest ibogaine interaction with opioid receptors, previous receptor binding studies (Brain Res. 571:242–247, 1980) found that ibogaine had a K_i value of only 2 μM for the kappa opioid receptor and was virtually inactive in blocking mu and delta receptor binding (K_i >100 μM). The present investigation of ibogaine interaction with the mu opioid receptor from mouse forebrain labeled with [3H]-naloxone, however, yielded significantly more potent mu opioid K_i values. LIGAND analysis indicated that the data were best fit by a two site binding model, with K_i values of about 130 nM and 4 μM, reflecting ibogaine recognition of different agonist affinity states of the receptor. Inclusion of 100 mM NaCl in the assay to induce the agonist low affinity state of the receptor, reduced ibogaine's inhibition of [³H]-naloxone binding. These results suggest that ibogaine is an agonist at the mu opioid receptor with a K_i value of about 130 nM, potentially explaining ibogaine's antinociceptive effects as well as its reported reduction of opioid withdrawal symptoms and attenuation of drug seeking behavior.     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

Effect of Iboga Alkaloids on μ-Opioid Receptor-Coupled G Protein Activation

Objective

The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR)-related G proteins by iboga alkaloids.

Methods

Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio)-triphosphate ([³⁵S]GTPγS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices.

Results And Significance

In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [³⁵S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [³⁵S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [³⁵S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.     

Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ～12 ? from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.