Alpha4beta2 nAChR Research Articles

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Understanding Nicotinic Receptor Assembly in the Endoplasmic Reticulum with Single Molecule Fluorescence Microscopy

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are expressed throughout the central and peripheral nervous systems. Some subtypes of these channels have been shown to upregulate in the presence of nicotine resulting in changes in expression levels and stoichiometry of receptors on the plasma membrane. We have developed a method capable of isolating intact and functional nAChRs from the endoplasmic reticulum using cell-derived vesicles. These nanoscale vesicles enable us to isolate single receptors in their native membrane providing a method to perform organelle specific single molecule stoichiometry measurements. I will explain the progress we have made using these methods to determine that nicotine and several other nAChR ligands alter the assembly of these receptors. Some ligands, including nicotine and its metabolites, lead to increased fractions of high sensitivity alpha4 beta2 nAChRs and others lead to higher fractions of the low sensitivity receptor. Additionally, many of these ligands lead to changes in receptor trafficking. Our results indicate that nAChR ligands act in the endoplasmic reticulum to assist in the assembly of nascent receptor subunits.

Of mice and Men; Neurobehavioral and toxicological effects of e-cigarettes exposure.

Event Abstract Back to Event Of mice and Men; Neurobehavioral and toxicological effects of e-cigarettes exposure. Tim Marczylo1*, Nathan Goldsmith1, 2, Elizabeth Zhuikova1, 2 and Alexis Bailey2 1 PHE, Toxicology, United Kingdom 2 St George's University of London, Institute of Medical and Biomedical Education, United Kingdom Electronic cigarettes (e-cigarettes) have proved very popular with smokers and a meteoric rise in their usage is currently under way. People purchase them as an aid to giving up smoking, to reduce cigarette consumption, to minimise withdrawal symptoms in environments that ban smoking, and in order to continue smoking with decreased health risks. Although the safety and impact on health of e-cigarettes, especially after long-term use, has not been evaluated, they are generally considered to be far safer alternatives to traditional tobacco cigarettes. This decrease in toxicity is predicted as a consequence of the absence or significantly reduced levels of the majority of toxicants in e-cigarette vape compared to tobacco smoke. However, there is evidence that the presence of nicotine in nicotine replacement therapies (NRT) can still generate carcinogenic tobacco-specific nitrosamines, albeit at very low concentrations in comparison with tobacco smoke. These nitrosamines are oesophageal and pulmonary carcinogens therefore nicotine delivery to the lung by e-cigarettes may carry an additional risk than other NRT. Additionally, nicotine itself is not without risk. Consequently, it is imperative to ascertain the toxicity risk of consuming nicotine through e-cigarettes. Our pilot study will monitor levels of nicotine, cotinine and carcinogenic tobacco-specific nitrosamines in the urine of heavy smokers (>10 cigarettes/day) who give up smoking and completely transition to e-cigarette use for a period of 4 weeks. Levels of other biomarkers which are known to be associated with smoking toxicity, such as DNA adducts and DNA methylation, will also be monitored in biological fluids of these subjects. Moreover, cigarette craving, mood, anxiety, social anxiety, well-being status and stress hormones (cortisol, oxytocin) will be measured throughout the transition from cigarette to e-cigarette use for 4 weeks to assess the psychological effect of the transition. Finally, brain electrical activity will be measured by electroencephalography pre- and post- transition to e-cigarette use. Study compliance will be monitored by expired carbon monoxide and urinary anabasine. A parallel study will be conducted in mice whereby nose only exposure to e-cigarette vape for a period of 90 days will be assessed for the aforementioned markers. Additionally, quantitative receptor autoradiography will be carried out in brains of these animals to investigate the effect of exposure to e-cigarette vapour on NMDA glutamate receptors, dopamine and nicotinic acetylcholine receptors (e.g. alpha4beta2 nAChRs) and cholinergic transporters in different brain regions. Overall, the results from these studies will provide important information on the safety and effectiveness of e-cigarettes for smoking cessation which we anticipate will drive policy decisions with respect to e-cigarettes and their use. Keywords: Smoking Cessation, electronic cigarettes, EEG, Nitrosamines, smokefreebrain Conference: SAN2016 Meeting, Corfu, Greece, 6 Oct - 9 Oct, 2016. Presentation Type: Oral Presentation in SAN 2016 Conference Topic: Oral Presentations Citation: Marczylo T, Goldsmith N, Zhuikova E and Bailey A (2016). Of mice and Men; Neurobehavioral and toxicological effects of e-cigarettes exposure.. Conference Abstract: SAN2016 Meeting. doi: 10.3389/conf.fnhum.2016.220.00019 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Jul 2016; Published Online: 30 Jul 2016. * Correspondence: Dr. Tim Marczylo, PHE, Toxicology, Oxford, Please select one, OX11 0RQ, United Kingdom, tim.marczylo@phe.gov.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Tim Marczylo Nathan Goldsmith Elizabeth Zhuikova Alexis Bailey Google Tim Marczylo Nathan Goldsmith Elizabeth Zhuikova Alexis Bailey Google Scholar Tim Marczylo Nathan Goldsmith Elizabeth Zhuikova Alexis Bailey PubMed Tim Marczylo Nathan Goldsmith Elizabeth Zhuikova Alexis Bailey Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Вплив агоніста та антагоніста α4β2 субодиниць Н-холінорецепторів на відтворення умовного рефлексу самиць щурів у різні фази естрального циклу

The present work was devoted to the comparative analysis of alpha4beta2 nicotinic acetylcholine receptors (nAChRs) in learning/ memory processes during ovary cycle assessed in adult female rats. RJR-2403 (1.0 mg/kg, i.p.), alpha4beta2 nAChRs agonist and mecamylamine (1.0 mg/kg, i.p.), alpha4beta2 nAChRs antagonist were injected chronically during 14 days. The processes of learning/memory were assessed in different models of learning: passive avoidance performance and Morris water maze. Chronic RJR-2403 administration to female rats improved the passive avoidance performance in proestrous and estrous as compared to the control animals. Also, RJR-2403 restored spatial learning during proestrous phases in Morris water maze, and stimulated the dynamics of spatial learning during estrous phases. On the contrary, chronic mecamylamine administration impaired non-spatial, and especially, spatial learning in females during key phases of ovary cycle. The results of the study suggest positive effect of alpha4beta2 nAChRs stimulation in learning/ memory processes during ovary cycle in the adult female rats.

The effects of noncontingent and self-administered cytisine on body weight and meal patterns in male Sprague–Dawley rats

RationaleIncreased appetite and weight gain after cessation are deterrents for quitting smoking. Pharmacotherapies that can reduce this weight gain in ex-smokers would be invaluable, and yet are not well studied in this context. ObjectiveTo examine the effects of extended daily exposure to intravenous cytisine, an alpha4beta2 nAChR partial agonist used for smoking cessation in some European countries, on body weight and patterns of food intake in rats. MethodsIn the first experiment, programmed infusions of cytisine were administered over 15h per day. Food intake, meal patterns, and weight change were examined relative to a vehicle-infused group during treatment, and in a post-cytisine phase. The second experiment examined the effects of cytisine on food intake, meal patterns, and weight change when substituted for nicotine in a self-administration protocol. Rats self-administered nicotine and cytisine during alternating four day periods, and changes in body weight, drug infusions, and meal patterns were compared between drugs and during an extinction phase. ResultsIn the first experiment, cytisine-treated rats ate less and gained less weight than those that received the vehicle. This occurred primarily by a reduced frequency of meals. In the 12day post-cytisine phase, animals maintained a lower body weight relative to controls throughout. In the second experiment, total pellet intake increased during cytisine substitution relative to nicotine and animals self-administered cytisine significantly less than nicotine. However, cytisine substitution maintained decreases in food intake and weight gain compared to baseline via decreases in total pellet intake and meal size. ConclusionCytisine administration results in decreased weight gain and changes in meal patterns dependent upon mode and pattern of administration and a previous history of nicotine administration.

Alexa Fluor 546‐ArIB[V11L;V16A] is a potent ligand for selectively labeling α7 nicotinic acetylcholine receptors

The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

Nicotinic acetylcholine receptors and depression: a review of the preclinical and clinical literature

Many patients with depression fail to derive sufficient benefit from available treatment options, with up to a third never reaching remission despite multiple trials of appropriate treatment. Novel antidepressant agents are needed, and drugs targeting nicotinic acetylcholine receptors (nAChRs) appear to hold promise in this regard. nAChRs are involved in a variety of neurobiological systems implicated in the pathophysiology of depression. In addition to their role in cholinergic neurotransmission, they modulate dopamine function and influence inflammation and hypothalamic-pituitary-adrenal axis activity. Preclinical studies have suggested antidepressant-like effects of drugs targeting nAChRs, with the most consistent results observed with alpha4beta2 nAChR modulators such as varenicline and nonspecific nAChR antagonists such as mecamylamine. These agents appear to offer the most potential antidepressant-like efficacy when used in conjunction with other established antidepressant treatments. nAChR modulators also influence neural processes that appear to mediate the behavioral effects of antidepressants, such as hippocampal cell proliferation. Clinical evidence, while limited, shows preliminary efficacy for mecamylamine and varenicline. Taken together, the preclinical and clinical evidence suggests that drugs targeting nAChRs may represent an important new approach to the treatment of depression.

Molecular mechanisms of cross‐inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK‐293 cells

P2X(2) and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and alpha3beta4subunit-containing nAChRs. The function of P2X2 and alpha3beta4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. In HEK-293 cells expressing alpha3beta4 nAChRs and P2X2 receptors, co-application of ATP and acetylcholine caused inward currents that were 56 +/- 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing alpha3beta4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing alpha3beta4 nAChRs transfected with P2X2TR. Currents caused by low (< or =30 micromol L(-1)), but not high (> =100 micromol L(-1)) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with alpha3beta4 nAChRs. The C-terminal tail of P2X2 receptors mediates cross-inhibition between alpha3beta4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross-inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters.

α-Conotoxin AuIB Isomers Exhibit Distinct Inhibitory Mechanisms and Differential Sensitivity to Stoichiometry of α3β4 Nicotinic Acetylcholine Receptors

Non-native disulfide isomers of alpha-conotoxins are generally inactive although some unexpectedly demonstrate comparable or enhanced bioactivity. The actions of "globular" and "ribbon" isomers of alpha-conotoxin AuIB have been characterized on alpha3beta4 nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes. Using two-electrode voltage clamp recording, we showed that the inhibitory efficacy of the ribbon isomer of AuIB is limited to approximately 50%. The maximal inhibition was stoichiometry-dependent because altering alpha3:beta4 RNA injection ratios either increased AuIB(ribbon) efficacy (10alpha:1beta) or completely abolished blockade (1alpha:10beta). In contrast, inhibition by AuIB(globular) was independent of injection ratios. ACh-evoked current amplitude was largest for 1:10 injected oocytes and smallest for the 10:1 ratio. ACh concentration-response curves revealed high (HS, 1:10) and low (LS, 10:1) sensitivity alpha3beta4 nAChRs with corresponding EC(50) values of 22.6 and 176.9 microM, respectively. Increasing the agonist concentration antagonized the inhibition of LS alpha3beta4 nAChRs by AuIB(ribbon), whereas inhibition of HS and LS alpha3beta4 nAChRs by AuIB(globular) was unaffected. Inhibition of LS and HS alpha3beta4 nAChRs by AuIB(globular) was insurmountable and independent of membrane potential. Molecular docking simulation suggested that AuIB(globular) is likely to bind to both alpha3beta4 nAChR stoichiometries outside of the ACh-binding pocket, whereas AuIB(ribbon) binds to the classical agonist-binding site of the LS alpha3beta4 nAChR only. In conclusion, the two isomers of AuIB differ in their inhibitory mechanisms such that AuIB(ribbon) inhibits only LS alpha3beta4 nAChRs competitively, whereas AuIB(globular) inhibits alpha3beta4 nAChRs irrespective of receptor stoichiometry, primarily by a non-competitive mechanism.

Negative Allosteric Modulators That Target Human α4β2 Neuronal Nicotinic Receptors

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human alpha4beta2 (Halpha4beta2) and human alpha3beta4 (Halpha3beta4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC(50) values ranging from 2.4 microM to more than 100 microM. Several NAMs showed relative selectivity for Halpha4beta2 nAChRs with IC(50) values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for Halpha4beta2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure-activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a Halpha4beta2 nAChR homology model, a binding mode for KAB-18 at the alpha/beta subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.

Pharmacological Characterization of the Allosteric Modulator Desformylflustrabromine and Its Interaction with α4β2 Neuronal Nicotinic Acetylcholine Receptor Orthosteric Ligands

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates alpha4beta2 nAChRs. It has been reported that dFBr is selective for the alpha4beta2 receptor relative to other common nAChR subtypes (Neurosci Lett 373:144-149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose-response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855-4860, 2007) at dFBr concentrations <10 microM and inhibition of responses at concentrations >10 microM. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human alpha4beta2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC(50) values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at alpha4beta2-containing synapses.

In Vitro Pharmacological Characterization of a Novel Selective α7 Neuronal Nicotinic Acetylcholine Receptor Agonist ABT-107

Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

Unresponsive Correlated Motion in α7 nAChR to Halothane Binding Explains Its Functional Insensitivity to Volatile Anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as targets for general anesthetics, but the functional responses to anesthetic modulation vary considerably among different subtypes of nAChRs. Inhaled general anesthetics, such as halothane, could effectively inhibit the channel activity of the alpha4beta2 nAChR but not the homologous alpha7 nAChR. To understand why alpha7 is insensitive to inhaled general anesthetics, we performed multiple sets of 20 ns molecular dynamics (MD) simulations on the closed- and open-channel alpha7 in the absence and presence of halothane and critically compared the results with those from our studies on the alpha4beta2 nAChR (Liu et al. J. Phys. Chem. B 2009, 113, 12581 and Liu et al. J. Phys. Chem. B 2010, 114, 626). Several halothane binding sites with fairly high binding affinities were identified in both closed- and open-channel alpha7, suggesting that a lack of sensitive functional responses of the alpha7 nAChR to halothane in the previous experiments was unlikely due to a lack of halothane interaction with alpha7. The binding affinities of halothane in alpha7 seemed to be protein conformation-dependent. Overall, halothane affinity was higher in the closed-channel alpha7. Halothane binding to alpha7 did not induce profound changes in alpha7 structure and dynamics that could be related to the channel function. In contrast, correlated motion of the open-channel alpha4beta2 was reduced substantially in the presence of halothane, primarily due to the more susceptible nature of beta2 to anesthetic modulation. The amphiphilic extracellular and transmembrane domain interface of the beta2 subunit is attractive to halothane and is susceptible to halothane perturbation, which may be why alpha4beta2 is functionally more sensitive to halothane than alpha7.

A Comparative Study of the Effects of the Intravenous Self-Administration or Subcutaneous Minipump Infusion of Nicotine on the Expression of Brain Neuronal Nicotinic Receptor Subtypes

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, alpha4beta2 nAChRs were up-regulated in all brain regions, alpha6beta2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and alpha7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of alpha4beta2alpha5 nAChRs in the CCx was also suggested. In the SA group, alpha4beta2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in alpha6beta2* or alpha7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in alpha4beta2 binding and alpha4 and beta2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in alpha4beta2 proteins. Western blotting also showed that the increase in the beta2 subunit exceeded that of the alpha4 subunit, suggesting that a change in alpha4beta2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor

Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric alpha7 nAChRs to heteromeric alpha3beta4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both alpha3 and beta4 subunits which, in order to monitor surface expression, were modified to bind alpha-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both alpha3 and beta4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the alpha3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the beta4 subunit. We also show that mutations at these domains, particularly loop 2 of the alpha3 subunit, change the pharmacological profile of alpha3beta4 nAChRs, decreasing nicotine's and increasing cytisine's effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-alpha subunits play a relevant role in the coupling process of neuronal alpha3beta4 nAChRs.

Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor

Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.

Varenicline does not increase serum BDNF levels in patients with nicotine dependence

Varenicline, alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist, is a new class of medications for treating nicotine dependence. As an alpha4beta2 nAChR partial agonist, varenicline serves to reduce nicotine withdrawal symptoms, while high-affinity binding of the agonist mitigates the reinforcing effects of smoking. In the present study, we compared serum brain-derived neurotrophic factor (BDNF) levels of nicotine dependence and nonsmokers, and we investigated changes in serum BDNF levels after 8 weeks of treatment with varenicline. Patients met the DSM-IV criteria for nicotine dependence. Both the Fagerström test for nicotine dependence (FTND) and the Tobacco Dependence Screener (TDS) were used. Serum BDNF levels and breath carbon monoxide (CO) levels were measured before and 8 weeks after varenicline treatment. Fourteen of 16 subjects (87.5%) stopped smoking within 12 weeks of varenicline treatment. Thirteen healthy nonsmokers who never had previously smoked were randomly selected as a control group. Serum BDNF levels of patients before treatment (4.8 +/- 3.8 ng/ml) were significantly lower than those in the control group (12.4 +/- 6.13 ng/ml). Serum BDNF levels had not increased from baseline (4.8 +/- 3.8 ng/ml) to 8 weeks after varenicline treatment (3.0 +/- 1.1 ng/ml) of patients. These results suggest that smoking might decrease serum BDNF levels and that treatment with varenicline for 8 weeks, combined with 12 weeks of not smoking, does not increase serum BDNF levels in smokers.

Nicotine provokes impulsive-like action by stimulating α4β2 nicotinic acetylcholine receptors in the infralimbic, but not in the prelimbic cortex

Nicotine, a major addictive component of tobacco, has been suggested to provoke impulsivity by activating central alpha4beta2 nicotinic acetylcholine receptors (nAChRs). Although lesion studies have demonstrated the involvement of the medial prefrontal cortex (mPFC) in impulsive action, the precise brain sites responsible for nicotine-induced impulsive action have not been identified. Our goal was to determine whether alpha4beta2 nAChRs in the prelimbic cortex (PL) and/or infralimbic cortex (IL), which are subregions of the mPFC, mediate nicotine-induced impulsive-like action in the three-choice serial reaction time task (3-CSRTT). The 3-CSRTT is a simple version of five-choice serial reaction time task and a rodent model of impulsive action in which the animal is required to inhibit the response until a light stimulus is presented randomly in one of three holes. Following the completion of the training, rats were bilaterally injected with dihydro-beta-erythroidine (DHbetaE; 6 and 18 microg/side), a selective alpha4beta2 nAChRs antagonist, into the PL or IL before systemic injection of nicotine (0.2 mg/kg, salt, s.c.). Intra-IL DHbetaE infusions dose-dependently blocked nicotine-induced impulsive-like action, while infusions of DHbetaE into the PL failed to block the effects of nicotine on impulsive-like action. The present results suggest a critical role for alpha4beta2 nAChRs in the IL in mediating the effects of nicotine on impulsive-like action in the 3-CSRTT.

Synthesis and Biological Evaluation of Bupropion Analogues as Potential Pharmacotherapies for Smoking Cessation

Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to antagonize the effects of human alpha3beta4*, alpha4beta2, alpha4beta4, and alpha1* nAChRs. The analogues were evaluated for their ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human alpha3beta4*-nAChR. Nine analogues have higher affinity at alpha3beta4*-nAChRs than 2a. Four analogues also had higher affinity for alpha4beta2 nAChR. Analogues 2r, 2m, and 2n with AD(50) values of 0.014, 0.015, and 0.028 mg/kg were 87, 81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test. Analogue 2x with IC(50) values of 31 and 180 nM for DA and NE, respectively, and with IC(50) of 0.62 and 9.8 microm for antagonism of alpha3beta4 and alpha4beta2 nAChRs had the best overall in vitro profile relative to 2a.

Enhancement of Attentional Performance by Selective Stimulation of α4β2* nAChRs: Underlying Cholinergic Mechanisms

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective alpha4beta2(*) nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity ('transients') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the alpha4beta2(*) nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the alpha7 nAChR 'sharpens' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the alpha7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, alpha4beta2(*) nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance.