Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1ß2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3ß2 â‰… α3ß4, indicating that ß2/ß4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9', 13', and 16', whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (-) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABAA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG ∼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.

DM506 (3-Methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a Novel Derivative of Ibogamine, Inhibits α7 and α9α10 Nicotinic Acetylcholine Receptors by Different Allosteric Mechanisms.

The main objective of this study was to determine the pharmacological activity and molecular mechanism of action of DM506 (3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a novel ibogamine derivative, at different nicotinic acetylcholine receptor (nAChR) subtypes. The functional results showed that DM506 neither activates nor potentiates but inhibits ACh-evoked currents at each rat nAChR subtype in a non-competitive manner. The receptor selectivity for DM506 inhibition follows the sequence: α9α10 (IC50 = 5.1 ± 0.3 µM) ≅ α7ß2 (5.6 ± 0.2 µM) ∼ α7 (6.4 ± 0.5 µM) > α6/α3ß2ß3 (25 ± 1 µM) > α4ß2 (62 ± 4 µM) ≅ α3ß4 (70 ± 5 µM). No significance differences in DM506 potency were observed between rat and human α7 and α9α10 nAChRs. These results also indicated that the ß2 subunit is not involved or is less relevant in the activity of DM506 at the α7ß2 nAChR. DM506 inhibits the α7 and α9α10 nAChRs in a voltage-dependent and voltage-independent manner, respectively. Molecular docking and molecular dynamics studies showed that DM506 forms stable interactions with a putative site located in the α7 cytoplasmic domain and with two intersubunit sites in the extracellular-transmembrane junction of the α9α10 nAChR, one located in the α10(+)/α10(─) interface and another in the α10(+)/α9(─) interface. This study shows for the first time that DM506 inhibits both α9α10 and α7 nAChR subtypes by novel allosteric mechanisms likely involving modulation of the extracellular-transmembrane domain junction and cytoplasmic domain, respectively, but not by direct competitive antagonism or open channel block.

Mutational Analysis of Anesthetic Binding Sites and Their Effects on GABAA Receptor Activation and Modulation by Positive Allosteric Modulators of the α7 Nicotinic Receptor.

The positive allosteric modulators (PAMs) of the α7 nicotinic receptor N-(5-Cl-2-hydroxyphenyl)-N'-[2-Cl-5-(trifluoromethyl)phenyl]-urea (NS-1738) and (E)-3-(furan-2-yl)-N-(p-tolyl)-acrylamide (PAM-2) potentiate the α1ß2γ2L GABAA receptor through interactions with the classic anesthetic binding sites located at intersubunit interfaces in the transmembrane domain of the receptor. In the present study, we employed mutational analysis to investigate in detail the involvement and contributions made by the individual intersubunit interfaces to receptor modulation by NS-1738 and PAM-2. We show that mutations to each of the anesthetic-binding intersubunit interfaces (ß+/α-, α+/ß-, and γ+/ß-), as well as the orphan α+/γ- interface, modify receptor potentiation by NS-1738 and PAM-2. Furthermore, mutations to any single interface can fully abolish potentiation by the α7-PAMs. The findings are discussed in the context of energetic additivity and interactions between the individual binding sites.

Modulation of the mammalian GABAA receptor by type I and type II positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

BACKGROUND AND PURPOSE: Positive allosteric modulators of the α7 nicotinic acetylcholine (nACh) receptor (α7-PAMs) possess promnesic and procognitive properties and have potential in the treatment of cognitive and psychiatric disorders including Alzheimer's disease and schizophrenia. Behavioural studies in rodents have indicated that α7-PAMs can also produce antinociceptive and anxiolytic effects that may be associated with positive modulation of the GABAA receptor. The overall goal of this study was to investigate the modulatory actions of selected α7-PAMs on the GABAA receptor. EXPERIMENTAL APPROACH: We employed a combination of cell fluorescence imaging, electrophysiology, functional competition and site-directed mutagenesis to investigate the functional and structural mechanisms of modulation of the GABAA receptor by three representative α7-PAMs. KEY RESULTS: We show that the α7-PAMs at micromolar concentrations enhance the apparent affinity of the GABAA receptor for the transmitter and potentiate current responses from the receptor. The compounds were equi-effective at binary αß and ternary αßγ GABAA receptors. Functional competition and site-directed mutagenesis indicate that the α7-PAMs bind to the classic anaesthetic binding sites in the transmembrane region in the intersubunit interfaces, which results in stabilization of the active state of the receptor. CONCLUSION AND IMPLICATIONS: We conclude that the tested α7-PAMs are micromolar-affinity, intermediate- to low-efficacy allosteric potentiators of the mammalian αßγ GABAA receptor. Given the similarities in the in vitro sensitivities of the α7 nACh and α1ß2γ2L GABAA receptors to α7-PAMs, we propose that doses used to produce nACh receptor-mediated behavioural effects in vivo are likely to modulate GABAA receptor function.

(E)-3-furan-2-yl-N-phenylacrylamide (PAM-4) decreases nociception and emotional manifestations of neuropathic pain in mice by α7 nicotinic acetylcholine receptor potentiation.

Clinical intervention of pain is often accompanied by changes in affective behaviors, so both assays of affective and sensorial aspects of nociception play an important role in the development of novel analgesics. Although positive allosteric modulation (PAM) of α7 nicotinic acetylcholine receptors (nAChRs) has been recognized as a novel approach for the relief of sensorial aspects of pain, their effects on affective components of pain remain unclear. Therefore, we investigated whether PAM-4, a highly selective α7-nAChR PAM, attenuates inflammatory and neuropathic pain, as well as the concomitant depressive/anxiety comorbidities. The anti-nociceptive activity of PAM-4 was assessed in mice using the formalin test and chronic constriction injury (CCI)-induced neuropathic pain model. The anxiolytic- and antidepressant-like activity of PAM-4 was evaluated using the marble burying test and forced swimming test. Acute systemic administration of PAM-4 dose-dependently reversed formalin-induced paw licking behavior and CCI-induced mechanical allodynia without development of any motor impairment. PAM-4 reversed the decreased swimming time and number of buried marbles in CCI-treated mice, suggesting that this ligand attenuates chronic pain-induced depression-like behavior and anxiogenic-like effects. The effects of PAM-4 were inhibited by the α7-selective antagonist methyllycaconitine, indicating molecular mechanism mediated by α7-nAChRs. Indeed, electrophysiological recordings showed the PAM-4 enhances human α7 nAChRs with higher potency and efficacy compared to rat α7 nAChRs. These findings suggest that PAM-4 reduces both sensorial and affective behaviors induced by chronic pain in mice by α7-nAChR potentiation. PAM-4 deserves further investigations for the management of chronic painful conditions with comorbidities.

Is the Antidepressant Activity of Selective Serotonin Reuptake Inhibitors Mediated by Nicotinic Acetylcholine Receptors?

It is generally assumed that selective serotonin reuptake inhibitors (SSRIs) induce antidepressant activity by inhibiting serotonin (5-HT) reuptake transporters, thus elevating synaptic 5-HT levels and, finally, ameliorates depression symptoms. New evidence indicates that SSRIs may also modulate other neurotransmitter systems by inhibiting neuronal nicotinic acetylcholine receptors (nAChRs), which are recognized as important in mood regulation. There is a clear and strong association between major depression and smoking, where depressed patients smoke twice as much as the normal population. However, SSRIs are not efficient for smoking cessation therapy. In patients with major depressive disorder, there is a lower availability of functional nAChRs, although their amount is not altered, which is possibly caused by higher endogenous ACh levels, which consequently induce nAChR desensitization. Other neurotransmitter systems have also emerged as possible targets for SSRIs. Studies on dorsal raphe nucleus serotoninergic neurons support the concept that SSRI-induced nAChR inhibition decreases the glutamatergic hyperstimulation observed in stress conditions, which compensates the excessive 5-HT overflow in these neurons and, consequently, ameliorates depression symptoms. At the molecular level, SSRIs inhibit different nAChR subtypes by noncompetitive mechanisms, including ion channel blockade and induction of receptor desensitization, whereas α9α10 nAChRs, which are peripherally expressed and not directly involved in depression, are inhibited by competitive mechanisms. According to the functional and structural results, SSRIs bind within the nAChR ion channel at high-affinity sites that are spread out between serine and valine rings. In conclusion, SSRI-induced inhibition of a variety of nAChRs expressed in different neurotransmitter systems widens the complexity by which these antidepressants may act clinically.

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3ß4* and α9α10 subtypes.

The inhibitory activity of (±)-citalopram on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3ß4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in µM) hα3ß4 AChRs (5.1 ±â€¯1.3) with higher potency than that for hα7 (18.8 ±â€¯1.1) and hα4ß2 (19.1 ±â€¯4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3ß4 with >2-fold higher affinity than that for hα4ß2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3ß4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3ß4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3ß4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

Alkaloids Purified from Aristotelia chilensis Inhibit the Human α3ß4 Nicotinic Acetylcholine Receptor with Higher Potencies Compared with the Human α4ß2 and α7 Subtypes.

The alkaloids aristoteline (1), aristoquinoline (2), and aristone (3) were purified from the leaves of the Maqui tree Aristotelia chilensis and chemically characterized by NMR spectroscopy. The pharmacological activity of these natural compounds was evaluated on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results suggest that these alkaloids do not have agonistic, but inhibitory, activity on each receptor subtype. The obtained IC50 values indicate the following receptor selectivity: hα3ß4 > hα4ß2 ≫ hα7. In the particular case of hα3ß4 AChRs, 1 (0.40 ± 0.20 µM) and 2 (0.96 ± 0.38 µM) show higher potencies compared with 3 (167 ± 3 µM). Molecular docking and structure-activity relationship results indicate that ligand lipophilicity is important for the interaction with the luminal site located close to the cytoplasmic side of the hα3ß4 ion channel between positions -2' and -4'. Compound 1 could be used as a molecular scaffold for the development of more potent noncompetitive inhibitors with higher selectivity for the hα3ß4 AChR that could serve for novel addiction and depression therapies.

Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in µM) rank: amitriptyline (2.7 ±â€¯0.3) > doxepin (5.9 ±â€¯1.1) ∼ imipramine (6.6 ±â€¯1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ±â€¯8.5 µM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ±â€¯0.05 µM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in µM): imipramine (1.6 ±â€¯0.2) > amitriptyline (2.4 ±â€¯0.3) > doxepin (4.9 ±â€¯0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action.

Drimane Sesquiterpenoids Noncompetitively Inhibit Human α4ß2 Nicotinic Acetylcholine Receptors with Higher Potency Compared to Human α3ß4 and α7 Subtypes.

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree ( Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4ß2, hα3ß4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4ß2 > hα3ß4 > hα7. In the case of hα4ß2 AChRs, the following potency rank order was determined (IC50's in µM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) ≫ dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4ß2 AChRs, additional structure-activity relationship and molecular docking experiments were performed. The Ca2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-ß2 intrasubunit) sites. The structure-activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4ß2 AChR by a cooperative mechanism, as shown experimentally ( nH > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4ß2 AChR.

Bupropion and its photoreactive analog (±)-SADU-3-72 interact with luminal and non-luminal sites at human α4ß2 nicotinic acetylcholine receptors.

The interaction of (±)-bupropion [(±)-BP] with the human (h) α4ß2 nicotinic acetylcholine receptor (AChR) was compared to that for its photoreactive analog (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72]. Ca2+ influx results indicated that (±)-SADU-3-72 and (±)-BP inhibit hα4ß2 AChRs with practically the same potency. However, (±)-SADU-3-72 binds to the [3H]imipramine sites at resting and desensitized hα4ß2 AChRs with 3-fold higher affinity compared to that for (±)-BP, which is supported by molecular docking results. The docking results also indicate that each isomer of BP and SADU-3-72, in the protonated state, interacts with luminal and non-luminal sites. In the channel lumen, both ligands bind to two overlapping subsites, one that overlaps the imipramine site, and another much closer to the cytoplasmic side. The results suggest, for the first time, three differentiated non-luminal domains, including the transmembrane (TMD), extracellular (ECD), and ECD-TMD junction. In the ECD-TMD junction, BP and SADU-3-72 bind to overlapping sites. Interestingly, only SADU-3-72 binds to intrasubunit and intersubunit sites in the TMD, and to additional sites in the ECD. Our results are consistent with a model where BP and SADU-3-72 bind to overlapping sites in the luminal and ECD-TMD junctional domains of the hα4ß2, whereas only SADU-3-72 binds to additional non-luminal sites. The BP junctional site opens the door for additional inhibitory mechanisms. The pharmacological properties of (±)-SADU-3-72 showed in this work support further photolabeling studies to mapping the BP binding sites in the hα4ß2 AChR.

(-)-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 Ca(2+) influx in human (h) muscle embryonic (hα1ß1γδ) and adult (hα1ß1εδ) 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 [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]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.

Novel 2-(substituted benzyl)quinuclidines inhibit human α7 and α4ß2 nicotinic receptors by different mechanisms.

This work presents the design and synthesis of a series of novel 2-benzylquinuclidine derivatives, comprising 12 methiodide and 11 hydrochloride salts, and their structural and pharmacological characterization at the human (h) α7 and α4ß2 nicotinic receptors (nAChRs). The antagonistic potency of these compounds was tested by Ca(2+) influx assays on cells expressing the hα7 or hα4ß2 nAChR subtype. To determine the inhibitory mechanisms, additional radioligand binding experiments were performed. The results indicate that the methiodides present the highest affinities for the hα7 nAChR agonist sites, while the same compounds bind preferably to the hα4ß2 nAChR ion channel domain. These results indicate that the methiodides are competitive antagonists of the hα7 nAChR but noncompetitive antagonists of the hα4ß2 subtype. Docking and molecular dynamics simulations showed that the methiodide derivative 8d binds to the hα7 orthosteric binding sites by forming stable cation-π interactions between the quaternized quinulinuim moiety and the aromatic box in the receptor, whereas compounds 7j and 8j block the hα4ß2 AChR ion channel by interacting with a luminal domain formed between the serine (position 6') and valine (position 13') rings that overlaps the imipramine binding site.

Functional and structural interaction of (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor.

The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4ß2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4ß2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4ß2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4ß2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4ß2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4ß2 nAChRs.

Neuronal networks of nicotine addiction.

Nicotine is the main psychoactive substance present in tobacco, targeting neuronal nicotinic acetylcholine receptors. The main effects of nicotine associated with smoking are nicotinic receptor activation, desensitization, and upregulation, with the subsequent modulation of the mesocorticolimbic dopaminergic system. However, there is a lack of a comprehensive explanation of their roles that effectively makes clear how nicotine dependence might be established on those grounds. Receptor upregulation is an unusual effect for a drug of abuse, because theoretically this implies less need for drug consumption. Receptor upregulation and receptor desensitization are commonly viewed as opposite, homeostatic mechanisms. We here review the available information on smoking addiction, especially under a recently presented model of nicotine dependence. In this model both receptor upregulation and receptor desensitization are responsible for establishing a biochemical mechanism of nicotine dependence, which have an important role in starting and maintaining tobacco addiction.

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 h alpha1beta1 gammadelta AChR-induced Ca(2+) influx with higher potencies than dizocilpine. The potency of fluoxetine is increased approximately 10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]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-(3)H(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 [(3)H]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.

Tobacco addiction: a biochemical model of nicotine dependence.

Nicotine is the main psychoactive substance present in tobacco, targeting in the CNS the nicotinic acetylcholine receptors (nAChR). The main effects of nicotine associated with smoking are nAChR upregulation, nAChR desensitization and modulation of the dopaminergic system. However, there is a lack of a comprehensive explanation of their roles that effectively makes clear how nicotine dependence might be established on those grounds. Receptor upregulation is an unusual effect for a drug of abuse, because theoretically this implies less need for drug consumption. Receptor upregulation and receptor desensitization are commonly viewed as opposite, homeostatic mechanisms. We here analyze the available information under a model in which both receptor upregulation and receptor desensitization are responsible for establishing a mechanism of nicotine dependence, consequently having an important role in starting and maintaining tobacco addiction. We propose that negative feedbacks on dopamine release regulated by alpha4beta2 nAChRs are disrupted by nicotine. nAChR desensitization is the disrupting mechanism, while nAChR upregulation is the reinforcing process of nicotine dependence, which eventually initiates tobacco addiction. A conclusion of the model is that drugs used for smoking cessation should inhibit preferentially alpha4beta2 nAChRs and to have a low or null ability to upregulate nAChRs, as this characteristic allows the smoker to achieve downregulation without abstinence symptoms. A relationship between this hypothesis and smoking and schizophrenia is also discussed.

A model for the assembly of nicotinic receptors based on subunit-subunit interactions.

Neuronal ion-channels are complex multimeric proteins. Within a given family, the variability of their pharmacological responses depends on subunit composition and subunit arrangement. We report here that protein assembly in the pentameric nicotinic acetylcholine receptor family, the best characterized of all neuronal receptors, can be predicted using information derived from homology modeled surface to surface subunit interactions based on the atomic structure of a snail acetylcholine-binding protein. An empirical assembly model is able to establish both subunit stoichiometry and subunit arrangement of known neuronal and muscle nicotinic receptors. This contribution to the understanding of nicotinic receptor assembly and variability might be extended to other types of ion-channels.