Neuronal Nicotinic Acetylcholine Receptors Research Articles

Spontaneous epileptic seizures in transgenic rats harboring a human ADNFLE missense mutation in the β2-subunit of the nicotinic acetylcholine receptor

We generated a transgenic rat strain with a missense mutation in V286L (V286L-TG), in the gene encoding the neuronal nicotinic acetylcholine receptor β2 subunit (CHRNB2) found in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To confirm that V286L-TG rats exhibit seizures similar to those observed in humans, gene expression patterns and behavioral phenotypes were analyzed. In situ hybridization using a V286L Chrnb2-selective probe indicated that the transgene was expressed at higher levels in the cortex, hippocampus, and cerebellum of V286L-TG than wild-type littermates (non-TG). Spontaneous epileptic seizures with ictal discharges in electroencephalograms were detected in 45% of V286L-TG rats and the frequency of seizures was 0.73 times a week. This seizure type is similar to “paroxysmal arousals” that are observed in human ADNFLE. V286L-TG rats displayed nicotine-induced abnormal motor activity including seizures in comparison to non-TGs. Response time following nicotine administration occurred faster in V286L-TG than in non-TG rats.V286L-TG rats demonstrated spontaneous epileptic seizures, which are similar to human ADNFLE, and also showed a higher sensitivity to nicotine administration. Thus, the V286L-TG rat model could be a valuable tool for developing novel mechanism-driven treatment strategies for epilepsy and provide a better understanding of ADNFLE.

Nicotinic modulation of auditory evoked potential electroencephalography in a rodent neurodevelopmental model of schizophrenia

Schizophrenia is a chronic disease that has been hypothesized to be linked to neurodevelopmental abnormalities. Schizophrenia patients exhibit impairments in basic sensory processing including sensory gating deficits in P50 and mismatch negativity (MMN). Neuronal nicotinic acetylcholine receptor (nAChR) agonists have been reported to attenuate these deficits. Gestational exposure of rats to methylazoxymethanol acetate (MAM) at embryonic day 17 leads to developmental disruption of the limbic-cortical system. MAM exposed offspring show neuropathological and behavioral changes that have similarities with those seen in schizophrenia. In this study, we aimed to assess whether N40 auditory sensory gating (the rodent form of P50 gating) and MMN deficits as measures of auditory evoked potential (AEP) electroencephalography (EEG) are present in MAM rats and whether nAChR agonists could attend the deficit. E17 male MAM and sham rats were implanted with cortical electrodes at 2 months of age. EEG recordings evaluating N40 gating and MMN paradigms were done comparing effects of vehicle (saline), nicotine and the α7 agonist ABT-107. Deficits were seen for MAM rats compared to sham animals in both N40 auditory sensory gating and MMN AEP recordings. There was a strong trend for N40 deficits to be attenuated by both nicotine (0.16mg/kg i.p. base) and ABT-107 (1.0mg/kg i.p. base). MMN deficits were significantly attenuated by ABT-107 but not by nicotine. These data support the MAM model as a useful tool for translating pharmacodynamic effects in clinical medicine studies of novel therapeutic treatments for schizophrenia.

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes.

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH2), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 310-helix from residues Pro7 to His10 and a type I β-turn from residues Pro14 to Cys17. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC50 of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu2 by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser12 with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.

Is postoperative delirium a relevant outcome?

potentially confounding neurological dysfunctions. In their study with 100 postoperative patients without a known risk factor of POD, the incidence of POD diagnosed using CAM was 28 %. Their result suggested that the incidence of POD was common in patients after major surgery even without known risk factors. Major surgery might evoke a strong stress response. Recent studies provided the hypothesis that it may introduce postoperative delirium or cognitive dysfunction [9, 10]. As the authors have pointed out, prior studies have commonly been conducted in postoperative patients with known risk factors; their study is relevant and novel. Another finding was a significant association of the use of thiopental with the risk of POD compared with propofol. Thiopental, a barbiturate class of drug, has effects on the gamma-aminobutyric acid (GABA) receptor [11] and propofol is also considered to act through potentiation of GABA receptor activity. Although their finding on thiopental may be skewed by the bias of patient characteristics and conditions which may cause anesthesiologists to choose thiopental rather than propofol, the potential action of thiopental on non-GABA-ergic ligand-gated ion channels, including the neuronal nicotinic acetylcholine receptor channels, may contribute to its association [12]. Although there are potential hypotheses for the etiology and pathophysiology of POD including cholinergic inhabitation, serotonin deficiency, dopamine activation [13], GABA activity [14], and melatonin activity [15], they are not fully understood [16]. This is one of the reasons why there are still just a few strategies for preventing POD [17]; the use of dexmedetomidine in post-cardiac surgery patients [18], multicomponent interventions, and antipsychotics. In this regard, further study should be warranted to develop a detailed understanding and preventive strategy for these postoperative complications. Delirium is characterized by the acute onset of mental status or a fluctuating course and inattention and disorganized thinking or an altered level of consciousness [1], which is common in critically ill patients [2]. Recently, the diagnosis of delirium is recommended by using the Confusion Assessment Method for the intensive care unit (CAM-ICU) [3]. Postoperative delirium (POD) was defined as so because it occurs postoperatively. Although operation, anesthetics, and analgesics can contribute to POD, it is not just associated with emergence from anesthesia, but often occurs between postoperative days 1 and 3. Although POD is commonly recovered from within a short period of time [4], patients with POD are more likely to die or develop dementia and require institutionalization [5]. A number of risk factors are reported for POD, including older age, preoperative cognitive or functional impairment, decreased postoperative hemoglobin, markedly abnormal sodium potassium and glucose, the presence of alcohol abuse, noncardiac thoracic surgery, aortic aneurysm surgery, history of delirium, preoperative use of narcotics, low postoperative oxygen saturation, history of cerebrovascular disease, and untreated pain [6, 7]. In a recent prospective observational study conducted by Saporito and Sturini [8], they excluded patients with any known preexisting predisposing factor for POD; types of surgery, preoperative dementia, sensory deficits, requirement of psychotropic drugs, and alcoholism etc., and other

Targeting brain nicotinic acetylcholine receptors to treat major depression and co-morbid alcohol or nicotine addiction.

Major depressive disorder (MDD) is a serious mental illness that affects millions of people worldwide. There is now compelling evidence that the neuronal nicotinic acetylcholine receptors (nAChRs) play an important role in MDD and co-morbid alcohol or nicotine addiction. As a result, there has been growing interest for the treatment of MDD and co-morbid alcohol or nicotine use disorder by targeting nAChRs. Emerging evidence suggests that specific ligands that act at nAChRs significantly reduce depression-like behaviors in preclinical models that mimic MDD and co-morbid alcohol or nicotine use disorder. In this review, the pharmacological efficacy of nAChR ligands, such as mecamylamine, lobeline, cytisine, sazetidine-A, and others will be discussed. Overall, findings from preclinical and clinical studies included here suggest that the nAChR ligands may be of potential benefit in reducing MDD symptoms and that may aid in the prevention and treatment of MDD and co-morbid alcohol or nicotine use disorder.

Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3–M4 cytoplasmic loop of the α4 subunit

The α4β2 neuronal nicotinic acetylcholine receptor (nAChR) plays a crucial role in nicotine addiction. These receptors are known to desensitize and up-regulate after chronic nicotine exposure, but the mechanism remains unknown. Currently, the structure and functional role of the intracellular domains of the nAChR are obscure. To study the effect of subunit phosphorylation on α4β2 nAChR function and expression, eleven residues located in the M3–M4 cytoplasmic loop were mutated to alanine and aspartic acid. Two-electrode voltage clamp and 125I-labeled epibatidine binding assays were performed on Xenopus oocytes to assess agonist activation and receptor expression. When ACh was used as an agonist, a decrease in receptor activation was observed for the majority of the mutations. When nicotine was used as an agonist, four mutations exhibited a statistically significant hypersensitivity to nicotine (S438D, S469A, Y576A, and S589A). Additionally, two mutations (S516D and T536A) that displayed normal activation with ACh displayed remarkable reductions in sensitivity to nicotine. Binding assays revealed a constitutive up-regulation in these two nicotine mutations with reduced nicotine sensitivity. These results suggest that consensus phosphorylation residues in the M3–M4 cytoplasmic loop of the α4 subunit play a crucial role in regulating α4β2 nAChR agonist selectivity and functional expression. Furthermore, these results suggest that disruption of specific interactions at PKC putative consensus sites can render α4β2 nAChRs almost insensitive to nicotine without substantial effects on normal AChR function. Therefore, these PKC consensus sites in the M3–M4 cytoplasmic loop of the α4 nAChR subunit could be a target for smoking cessation drugs.

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.

Radiosynthesis and in vitro validation of 3H-NS14492 as a novel high affinity alpha7 nicotinic receptor radioligand

The neuronal α7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer׳s disease and schizophrenia. We have previously described 11C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the α7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. 3H-NS14492 showed specific binding to α7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1±0.7nM and a maximum number of binding sites (Bmax) of 15.7±2.0fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand 125I-α-bungarotoxin (125I-BTX) in autoradiography, and unlabeled NS14492 displaced 125I-BTX with an inhibition constant (Ki) of 23nM. 3H-NS14492 bound to α7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3nM and a Bmax of 30.2±11.6fmol/mg protein. This binding assay further revealed the Ki rank order for a number of α7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of 3H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that 3H-NS14492 is a useful new in vitro radioligand for the pig α7 nicotinic receptor.

In vitro and in vivo neuronal nicotinic receptor properties of (+)- and (-)-pyrido[3,4]homotropane [(+)- and (-)-PHT]: (+)-PHT is a potent and selective full agonist at α6β2 containing neuronal nicotinic acetylcholine receptors.

Pyrido[3,4]homotropane (PHT) is a conformationally rigid, high affinity analogue of nicotine. (+)-PHT was previously shown to be 266 times more potent than (-)-PHT for inhibition of [(3)H]epibatidine binding to nAChRs but had no antinociceptive activity in mouse tail-flick or hot-plate tests and was not a nicotinic antagonist even when administered intrathecally. While (-)-PHT had no agonist activity, it was a potent, nicotinic antagonist in the test. Here, electrophysiological studies with rat nAChRs show (+)-PHT to be a low efficacy partial agonist selective for α4β2-nAChRs, relative to α3β4-nAChRs (15-fold) and α7-nAChRs (45-fold). (-)-PHT was an antagonist with selectivity for α3β4, relative to α4β2- (3-fold) and α7- (11-fold) nAChRs. In [(3)H]DA release studies in mice, (+)-PHT was 10-fold more potent than (-)-PHT at α4β2*-nAChRs and 30-fold more potent at α6β2*-nAChRs. Studies using α5KO mice suggested that much of the activity at α4β2*-nAChRs is mediated by the α4β2α5-nAChR subtype. In conditioned place preference studies, (-)-PHT was more potent than (+)-PHT in blocking nicotine reward. Off-target screens showed (+)- and (-)-PHT to be highly selective for nAChRs. The high potency, full agonism of (+)- and (-)-PHT at α6*-nAChR contrasts with the partial agonism observed for α4*-nAChR, making these ligands intriguing probes for learning more about the pharmacophores for various nAChRs.

Crucial role of nicotinic α5 subunit variants for Ca2+ fluxes in ventral midbrain neurons.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (α5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the α5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca(2+) concentration elevations and that in α5(-/-) mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the α5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how α5DN impairs heteromeric nAChR functions, we coexpressed α4, α5, or α5DN subunits with a dimeric concatemer (β2α4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both α5(β2α4)2 and α5DN(β2α4)2 nAChRs yielded similar levels of functional expression and Ca(2+) permeability, measured as fractional Ca(2+) currents (8.2 ± 0.7% and 8.0 ± 1.9%, respectively), 2-fold higher than α4(β2α4)2. Our results indicate that the loss of function of nicotinic responses observed in α5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca(2+) permeability but likely to intracellular modulation.

Characterization of Noribogaine at nAChRs and Effect on Nicotine Self‐Administration in Rats

Noribogaine, a polypharmacological drug with activities at opioid receptors, ionotropic nicotinic receptors, and serotonin reuptake transporters, is being investigated for treatment of substance abuse‐related disorders. In this study, noribogaine molecular activity at neuronal nicotinic acetylcholine receptors (nAChRs) was characterized. Noribogaine inhibited α3β4, α7 nAChRs, and endogenously expressed habenula‐type α3‐(α5, β2, β4) nAChRs populations. Anti‐addictive compounds ibogaine, 18MC‐ a synthetic derivative of coronaridine, and mecamylamine were tested in parallel. Then, noribogaine was tested for use as a treatment for nicotine dependence in a nicotine self‐administration study in rats in comparison to varenicline, an approved medication for smoking cessation. After initial food pellet training, followed by 26 sessions of nicotine self‐administration training, the rats were administered noribogaine (12.5, 25 or 50 mg/kg p.o.), noribogaine vehicle, varenicline or saline using a within‐subject design with a latin square test schedule. Noribogaine dose‐dependently decreased nicotine self‐administration by up to 64% of saline treated rats levels and was equi‐effective to varenicline. Noribogaine pharmacological profiling at the nAChRs receptor population and its ability to dose‐dependently attenuate drug‐taking behavior for nicotine supports future studies to assess its potential usefulness in multiple therapeutic arenas, including smoking cessation, substance abuse related disorders, and anxiety related disorders.

Tying up Nicotine: New Selective Competitive Antagonist of the Neuronal Nicotinic Acetylcholine Receptors.

Conformational restriction of the pyrrolidine nitrogen in nicotine by the introduction of an ethylene bridge provided a potent and selective antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors. Resolution by chiral SFC, pharmacological characterization of the two enantiomers, and determination of absolute configuration via enantioselective synthesis showed that the pharmacological activity resided almost exclusively in the (R)-enantiomer.

Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility.

Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including autism spectrum disorder (ASD), seizures, intellectual disability, and other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the alpha7 subunit of the neuronal nicotinic acetylcholine receptor, is considered the likely culprit gene in mediating neurological phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare variants confer risk to ASD, we performed copy number variant analysis and Sanger sequencing of the CHRNA7 coding sequence in a sample of 135 ASD cases. Sequence variation in this gene remains largely unexplored, given the existence of a fusion gene, CHRFAM7A, which includes a nearly identical partial duplication of CHRNA7. Hence, attempts to sequence coding exons must distinguish between CHRNA7 and CHRFAM7A, making next-generation sequencing approaches unreliable for this purpose. A CHRNA7 microduplication was detected in a patient with autism and moderate cognitive impairment; while no rare damaging variants were identified in the coding region, we detected rare variants in the promoter region, previously described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample of idiopathic autism.

Pharmacological characterization of conotoxin lt14a as a potent non-addictive analgesic.

Conotoxin lt14a is a small peptide consisting of 13 amino acids. It was originally identified from the cDNA of Conus litteratus in the South China Sea. Previous reports showed lt14a exhibited antinociceptive activity using a hot plate-induced pain mouse model and acted as an antagonist of neuronal nicotinic acetylcholine receptors. We confirmed that conotoxin lt14a administration resulted in antinociception activity using a mouse inflammatory pain model and a rat model of mechanically-induced pain. The mRNA expression of c-fos and NOS in the spinal cord of rats was suppressed by lt14a. Labeling of lt14a with an Alexa Fluor 488 ester showed that lt14a was bound to the surface of PC12 cells and that this binding was inhibited by pre-application of the nicotinic acetylcholine receptor (nAChR) antagonist tubocurarine chloride (TUB) and the nAChR blocker hexamethonium bromide (HB). These data confirm previous reports that showed lt14a binds to the surface of PC12 cells via nAChRs with patch clamp whole-cell recordings. Additional results showed that lt14a suppressed extracellular signal-regulated kinase (ERK1/2) phosphorylation in PC12 cells activated by Ach. Our results showed that lt14a did not induce drug dependence but rather suppressed morphine withdrawal symptoms. Our work suggests that lt14a is a novel antinociceptive agent that targets the nAChR receptor without inducing drug dependence.

Preclinical studies of the potent and selective nicotinic α4β2 receptor ligand VMY-2-95.

The discovery and development of small molecules that antagonize neuronal nicotinic acetylcholine receptors may provide new ligands for evaluation in models of depression or addiction. We discovered a small molecule, VMY-2-95, a nAChR ligand with picomolar affinity and high selectivity for α4β2 receptors. In this study, we investigated its preclinical profile in regards to solubility, lipophilicity, metabolic stability, intestinal permeability, bioavailability, and drug delivery to the rat brain. Metabolic stability of VMY-2-95·2HCl was monitored on human liver microsomes, and specific activity of VMY-2-95·2HCl on substrate metabolism by CYP1A2, 2C9, 2C19, 2D6, and 3A4 was tested in a high-throughput manner. The intestinal transport of VMY-2-95·2HCl was studied through Caco-2 cell monolayer permeability. VMY-2-95·2HCl was soluble in water and chemically stable, and the apparent partition coefficient was 0.682. VMY-2-95·2HCl showed significant inhibition of CYP2C9 and 2C19, but weak or no effect on 1A2, 2D6, and 3A4. The Caco-2 cell model studies revealed that VMY-2-95·2HCl was highly permeable with efflux ratio of 1.11. VMY-2-95·2HCl achieved a maximum serum concentration of 0.56 mg/mL at 0.9 h and was orally available with a half-life of ∼9 h. Furthermore, VMY-2-95·2HCl was detected in the rat brain after 3 mg/kg oral administration and achieved a maximal brain tissue concentration of 2.3 μg/g within 60 min. Overall, the results demonstrate that VMY-2-95·2HCl has good drug like properties and can penetrate the blood–brain barrier with oral administration.

Differential expression of the beta4 neuronal nicotinic receptor subunit affects tolerance development and nicotinic binding sites following chronic nicotine treatment

The role of neuronal nicotinic acetylcholine receptors (nAChR) containing the β4 subunit in tolerance development and nicotinic binding site levels following chronic nicotine treatment was investigated. Mice differing in expression of the β4-nAChR subunit [wild-type (β4++), heterozygote (β4+−) and null mutant (β4−−)] were chronically treated for 10days with nicotine (0, 0.5, 1.0, 2.0 or 4.0mg/kg/h) by constant intravenous infusion. Chronic nicotine treatment elicited dose-dependent tolerance development. β4−− mice developed significantly more tolerance than either β4++ or β4+− mice which was most evident following treatment with 4.0mg/kg/h nicotine. Subsets of [125I]-epibatidine binding were measured in several brain regions. Deletion of the β4 subunit had little effect on initial levels of cytisine-sensitive [125I]-epibatidine binding (primarily α4β2-nAChR sites) or their response (generally increased binding) to chronic nicotine treatment. In contrast, β4 gene-dose-dependent decreases in expression 5IA-85380 resistant [125I]-epibatidine binding sites (primarily β4*-nAChR) were observed. While these β4*-nAChR sites were generally resistant to regulation by chronic nicotine treatment, significant increases in binding were noted for habenula and hindbrain. Comparison of previously published tolerance development in β2−− mice (less tolerance) to that of β4−− mice (more tolerance) supports a differential role for these receptor subtypes in regulating tolerance following chronic nicotine treatment.

Pathologic role of neuronal nicotinic acetylcholine receptors in epileptic disorders: implication for pharmacological interventions.

Accumulating evidence suggests that neuronal nicotinic acetylcholine receptors (nAChRs) may play a key role in the pathophysiology of some neurological diseases such as epilepsy. Based on genetic studies in patients with epileptic disorders worldwide and animal models of seizure, it has been demonstrated that nAChR activity is altered in some specific types of epilepsy, including autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and juvenile myoclonic epilepsy (JME). Neuronal nAChR antagonists also have antiepileptic effects in pre-clinical studies. There is some evidence that conventional antiepileptic drugs may affect neuronal nAChR function. In this review, we re-examine the evidence for the involvement of nAChRs in the pathophysiology of some epileptic disorders, especially ADNFLE and JME, and provide an overview of nAChR antagonists that have been evaluated in animal models of seizure.

Interaction of Bupropion with 5-HT3A Receptors

For more than two decades, bupropion has been clinically prescribed for the treatment of depression (Wellbutrin®), and more recently for smoking cessation (Zyban®). Bupropion is conventionally described as a dual norepinephrine dopamine reuptake inhibitor, therefore effective as an antidepressant. However, we and others have shown that it also interacts with receptors of the Cys-loop superfamily, namely neuronal nicotinic acetylcholine receptors. In the continuum of exploring the molecular interactions of bupropion with other prominent members of the Cys-loop superfamily, we report here that bupropion additionally inhibits 5-hydroxytryptamine (serotonin) type 3A receptors (5-HT3ARs).5-HT3ARs are cation-conducting, homo-pentameric ligand-gated ion channels of the Cys-loop superfamily which are structurally and functionally distinct from other serotonin receptors that are G-proteins. 5-HT3 receptors are current targets for anti-emetics mainly used in cancer chemotherapy, as well as potential future targets for disorders including anxiety, schizophrenia, and Alzheimer's disease.Here we characterized our newly-identified interaction of bupropion with 5-HT3ARs with electrophysiological and radioligand binding studies. In oocytes, bupropion inhibited 5-HT3AR-mediated currents with an IC50 value of 87 µM (nH = 1.2). That 300 µM bupropion inhibited [3H]-5-HT or [3H]-granisetron (a competitive antagonist) binding by ∼ 20% indicated at best weak interaction with the agonist binding site. In contrast, the effect of different concentrations of bupropion on serotonin concentration-response curves suggested a non-competitive nature of interaction. Along these lines, importantly, the inhibition of serotonin-evoked currents recorded from oocytes was proportional to the length of preincubation time with bupropion.In summary, our results are indicative of a negative allosteric interaction of bupropion with the 5-HT3AR. Currently, experiments are underway to identify the site(s) of interaction for bupropion within the 5- HT3AR.

Interaction of 7-Methoxytacrine-Adamantylamine Cholinesterase Inhibitors with Nicotinic and Muscarinic Acetylcholine Receptors

Acetylcholinesterase inhibitors (AChEI) and the N-methyl-D-aspartate (NMDA) receptor antagonist memantine are among the few FDA-approved drugs for Alzheimer's disease. In effort to develop multi-target (AChE-NMDA)-directed ligands for the treatment of Alzheimer's disease, a series 7-Methoxytacrine-Adamantylamine thiourea heterodimers have been synthesized and evaluated for their effects as AChEI (Spilovska et. al. 2013, Molecules 18, 2397-2418). In this report, we extend their pharmacological characterization by examining their effects on neuronal and muscle-type nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (mAChR). We also compare their pharmacology to 7-Methoxytacrine-Adamantylamine urea heterodimers. 7-Methoxytacrine-Adamantylamine thiourea derivatives containing 2-8 carbon linkers inhibited AChE and butyrylcholinesterase (BChE) with high potency (IC50, 0.5-6 μM), whereas 7-Methoxytacrine-Adamantylamine urea derivatives inhibited AChE with higher potency than BChE. None of the tested compound potentiated M1 mAChR or nAChRs responses. They have minimal effects on acetylcholine-induced currents in Xenopus oocytes expressing neuronal α4β2 nAChR and inhibited M1 mAChR at higher concentrations (IC50s >10 μM). At concentration that inhibit AChE, both thiourea and urea derivatives inhibited acetylcholine-induced currents in Xenopus oocytes expressing muscle-type AChRs. Our preliminary results also suggest that the length of the carbon linker affect 7-Methoxytacrine-Adamantylamine urea interactions with AChRs, and further modifications of their structure are expected to yield compounds with more favorable pharmacological profiles.

Organization and Dynamics of Nicotinic Acetylcholine Receptor Nanoclusters at the Cell Surface

Organization and maintenance of neurotransmitter receptor assemblies, key for efficient synaptic transmission, rely on an adequate balance of synthesis, delivery to and removal from the cell membrane. We are studying the supramolecular organization, dynamics and trafficking of neuronal and muscle-type nicotinic acetylcholine receptors (nAChRs) using a combination of ensemble averaging methods and single-molecule experimental techniques. STED and GSDIM microscopies (two forms of nanoscopy) of fixed specimens of a clonal cell line expressing muscle-type AChR disclosed diffraction barrier-breaking light microscopy images of ∼55 nm cell-surface particles (“nanoclusters”) which increase in size upon ligation. Live cell imaging has enabled the visualization of particles in real time, and single-particle tracking together with cluster analysis reveals the motional regimes and modes or supramolecular assembly of nAChR nanoclusters. The neutral lipid cholesterol and cortical actin dynamics act synergically to modulate the supramolecular organization and dynamics of nAChRs at the cell surface.Research quoted here was supported by grant PICT 2011-0604 from the Argentinian Ministry for Science, Technology and Productive Innovation to F.J.B.