nAChR Antagonist Research Articles

Scaffold Ranking and Positional Scanning Utilized in the Discovery of nAChR-Selective Compounds Suitable for Optimization Studies

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4β2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior. Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4β2 nAChR selective compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4β2 nAChR antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC50 of approximately 430 nM. 301 demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4β2 over α3β4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation medications.

SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205+ dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity

Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including α7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous α7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205+ dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10μg/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific α7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5μg/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at α7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses.

The alpha-7 nicotinic receptor partial agonist/5-HT3 antagonist RG3487 enhances cortical and hippocampal dopamine and acetylcholine release

Alpha-7 nicotinic acetylcholine receptor (nAChR) agonists may ameliorate cognitive deficits in schizophrenia, in part, because of their ability to enhance dopaminergic and cholinergic neurotransmission. In the current study, the effects of partial nAChR agonist and 5-HT3 receptor antagonist RG3487 (previously R3487/MEM3454) on dopamine (DA) and acetylcholine (ACh) effluxes in rat prefrontal cortex (mPFC) and hippocampus (HIP) were investigated in awake, freely moving rats. R3487/MEM3454, at doses of 0.1-10mg/kg, s.c., enhanced DA and ACh effluxes in rat mPFC and (HIP), with a peak effect at 0.3- to 0.6-mg/kg doses, producing a bell-shaped dose-response curve. Pretreatment with the selective nAChR antagonist, methyllycaconitine (1.0mg/kg), completely blocked RG3487-induced (0.45mg/kg) DA but not ACh efflux, while the selective 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (1.0mg/kg) partially inhibited cortical ACh but not DA efflux. RG3487 (0.45mg/kg) combined with atypical antipsychotic drug (APD) risperidone (0.1mg/kg), but not typical APD haloperidol (0.1mg/kg), induced a significantly greater increase in HIP ACh efflux. Their combined effect on DA efflux was additive. RG3487, combined with other atypical APDs, namely aripiprazole (0.3mg/kg), olanzapine (1.0mg/kg), and quetiapine (30mg/kg), also produced additive effects on DA efflux. These results suggest that RG3487 enhances DA efflux by nAChR stimulation, whereas ACh efflux is primarily mediated via 5-HT3 receptor antagonism, and that RG3487 alone or as augmentation may improve cognitive impairment in schizophrenia.

Role of neuronal nicotinic acetylcholine receptors (nAChRs) on learning and memory in zebrafish

Neuronal nicotinic acetylcholine receptors (nAChRs) play a modulatory role in cognition, and zebrafish provide a preclinical model to study learning and memory. We investigated the effect of nicotine (NIC) and some new cytisine-derived partial agonists (CC4 and CC26) on spatial memory in zebrafish using a rapid assay on T-maze task. The role of α4/α6β2 and the α7 nAChRs in NIC-induced memory enhancement was evaluated using selective nAChR antagonists. Low and high doses of NIC, cytisine (CYT), CC4 and CC26 respectively improved and worsened the mean running time, showing an inverted U dose-response function. The effective dose (ED50) (×10⁻⁵ mg/kg) was 0.4 for CC4, 4.5 for CYT, 140 for NIC and 200 for CC26. NIC-induced cognitive enhancement was reduced by the selective nAChR subtype antagonists: methyllycaconitine (MLA) for α7, α-conotoxin (MII) for α6β2, dihydro-β-erythroidine (DhβE) for α4β2, the nonselective antagonist mecamylamine (MEC) and the muscarinic antagonist scopolamine (SCOP), with DhβE being more active than MLA or MII. All the partial agonists blocked the cognitive enhancement. The improvement with the maximal active dose of each partial agonist was blocked by low doses of DhβE (0.001 mg/kg) and MII (0.01 mg/kg). MLA reduced the effects of CC26 and CC4 at doses of 0.01 and 1 mg/kg, respectively, but did not antagonize CYT-induced memory improvement at any of the tested dose. No change in swimming activity was observed. Our findings demonstrate that zebrafish make a useful model for the rapid screening of the effect of new α4β2 nAChR compounds on spatial memory.

Characterization of a Novel α-Conotoxin TxID from Conus textile That Potently Blocks Rat α3β4 Nicotinic Acetylcholine Receptors

The α3β4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an α-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods, and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat α3β4 nAChRs with a 12.5 nM IC50, which places it among the most potent α3β4 nAChR antagonists. TxID also blocked the closely related α6/α3β4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular α-CTx fold but with different surface charge distribution to other 4/6 family members. α-CTx TxID is a novel tool with which to probe the structure and function of α3β4 nAChRs.

Identifying Key Amino Acid Residues That Affect α-Conotoxin AuIB Inhibition of α3β4 Nicotinic Acetylcholine Receptors

α-Conotoxin AuIB is a selective α3β4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and α3β4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·α3β4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the α3β4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of α3β4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the β4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the β4-Trp-59 and β4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the β4 subunit and gave further insight into how AuIB Phe-9 interacts with the β4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.

Design, Synthesis, and Activity of a Series of Arylpyrid-3-ylmethanones as Type I Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors

A series of novel arylpyrid-3-ylmethanones (7a-aa) were designed as modulators of α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be type I positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes. Structure-activity relationship (SAR) studies resulted in the identification of compound 7v as a potent and efficacious type I PAM with maximum modulation of a nicotine EC5 response of 1200% and EC50 = 0.18 μM. Compound 7z was active in reversing the effect of scopolamine in the novel object recognition (NOR) paradigm with a minimum effective ip dose of 1.0 mg/kg (2.7 μmol/kg). This effect was blocked by the selective α7 nAChR antagonist methyllycaconitine (MLA). These compounds are potent type I positive allosteric modulators of α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive deficits in schizophrenia and Alzheimer's disease.

Nicotine induces dendritic spine remodeling in cultured hippocampal neurons

Cholinergic neurons in the CNS are involved in synaptic plasticity and cognition. Both muscarinic and nicotinic acetylcholine receptors (nAChRs) influence plasticity and cognitive function. The mechanism underlying nAChR-induced plasticity, however, has remained elusive. Here, we demonstrate morphological changes in dendritic spines following activation of α4β2* nAChRs, which are expressed on glutamatergic pre-synaptic termini of cultured hippocampal neurons. Exposure of the neurons to nicotine resulted in a lateral enlargement of spine heads. This was abolished by dihydro-β-erythroidine, an antagonist of α4β2* nAChRs, but not by α-bungarotoxin, an antagonist of α7 nAChRs. Tetanus toxin or a mixture of 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, antagonists of NMDA- and AMPA-type glutamate receptors, blocked the nicotine-induced spine remodeling. In addition, nicotine exerted full spine-enlarging response in the post-synaptic neuron whose β2 nAChR expression was knocked down. Finally, pre-treatment with nicotine enhanced the Ca(2+)-response of the neurons to glutamate. These data suggest that nicotine influences the activity of glutamatergic neurotransmission through the activation of pre-synaptic α4β2 nAChRs, resulting in the modulation of spinal architecture and responsiveness. The present findings may represent one of the cellular mechanisms underlying cholinergic tuning of brain function. Activation of nicotinic acetylcholine receptors (nAChRs) in brain influences plasticity and cognition. Here, activation of α4β2* nAChRs, which are expressed on glutamatergic presynaptic termini, results in the enlargement of dendritic spines through the modulation of the glutamatergic neurotransmission. The remodeled spinal architecture might be responsible for the change in responsiveness of neural circuitry, leading to cholinergic tuning of brain function.

Long‐term nicotine treatment down‐regulates α6β2* nicotinic receptor expression and function in nucleus accumbens

Long-term nicotine exposure induces alterations in dopamine transmission in nucleus accumbens that sustain the reinforcing effects of smoking. One approach to understand the adaptive changes that arise involves measurement of endogenous dopamine release using voltammetry. We therefore treated rats for 2-3 months with nicotine and examined alterations in nAChR subtype expression and electrically evoked dopamine release in rat nucleus accumbens shell, a region key in addiction. Long-term nicotine treatment selectively decreased stimulated α6β2* nAChR-mediated dopamine release compared with vehicle-treated rats. It also reduced α6β2* nAChRs, suggesting the receptor decline may contribute to the functional loss. This decreased response in release after chronic nicotine treatment was still partially sensitive to the agonist nicotine. Studies with an acetylcholinesterase inhibitor demonstrated that the response was also sensitive to increased endogenous acetylcholine. However, unlike the agonists, nAChR antagonists decreased dopamine release only in vehicle- but not nicotine-treated rats. As antagonists function by blocking the action of acetylcholine, their ineffectiveness suggests that reduced acetylcholine levels partly underlie the dampened α6β2* nAChR-mediated function in nicotine-treated rats. As long-term nicotine modifies dopamine release by decreasing α6β2* nAChRs and their function, these data suggest that interventions that target this subtype may be useful for treating nicotine dependence. Long-term nicotine treatment decreases dopamine (DA) transmission in the mesolimbic dopaminergic system. Our data suggest this may involve a decrease in α6β2* nicotinic receptor expression and function. These changes may play a key role in nicotine reward and dependence.

Acute effects of mecamylamine and varenicline on cognitive performance in non-smokers with and without schizophrenia.

Nicotinic acetylcholine receptors (nAChRs) have been implicated in the pathophysiology of cognitive deficits in the domains of attention and memory in schizophrenia. While nicotinic agonists and antagonists have been proposed as smoking cessation aids, few comparisons have been made of these agents on cognitive performance in individuals with schizophrenia. This study investigated the acute effects of a nAChR antagonist, mecamylamine, and partial agonist, varenicline, on cognitive function in non-smokers with and without schizophrenia. Single oral doses of mecamylamine 10 mg, varenicline 1 mg, and placebo were administered 1 week apart in random order to adults with schizophrenia (n = 30) and to healthy volunteers (n = 41) in a double-blind, crossover design. The primary outcome of interest was sustained attention as assessed with hit reaction time variability (HRT-SD) on the identical pairs continuous performance test (CPT-IP). Mecamylamine worsened performance on CPT-IP HRT-SD, a measure of attention, compared to varenicline in both groups. Performance on mecamylamine was worse than performance on both placebo and varenicline on several additional measures of attention, including CPT-IP hit reaction time (HRT) and random errors at various levels of task difficulty. There was a treatment by diagnosis interaction, such that mecamylamine worsened performance on CPT-IP 2-digit HRT, 3-digit random errors, and 4-digit hit rate compared to placebo and varenicline in participants with schizophrenia; effects not observed in controls. These findings support a role for nAChRs in attention and suggest that those with schizophrenia may be particularly sensitive to nAChR blockade.

Tyrosine phosphatase STEP61 negatively regulates amyloid β‐mediated ERK/CREB signaling pathways via α7 nicotinic acetylcholine receptors

Striatal-enriched phosphatase 61 (STEP61 ) plays an essential role in synaptic plasticity and has recently been implicated in neurodegenerative disease. Here we characterized a possible role of STEP61 in Alzheimer's disease (AD) pathology using a mouse model of AD (Tg-APPswe/PSEN1dE9, APP/PS1 mice) and an in vitro model of AD [cortical neurons treated with amyloid β (Aβ)1-42 peptides]. Our data indicate age-related elevation of STEP61 levels and the proportion of dephosphorylated STEP61 (active STEP61 ) in wild-type mice, which was enhanced in APP/PS1 mice. Furthermore, the increased STEP61 levels and active STEP61 were observed in the hippocampus and cortex from 12-month-old APP/PS1 mice and in Aβ1-42 -treated cortical neurons. An α7 nicotinic acetylcholine receptors (nAChRs) antagonist, α-bungarotoxin (BTX), inhibited the Aβ1-42 -induced increase of STEP61 expression and activation. In addition, extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP response element binding (CREB) were impaired in Aβ1-42 -treated cortical neurons, and knockdown of STEP61 enhanced the activation of ERK1/2 and CREB. Collectively, these findings indicate two alternate pathological pathways effecting STEP61 regulation in AD. First, Aβ regulating STEP61 activity is mediated by Aβ binding to α7 nAChRs. Second, STEP61 negatively regulates Aβ-mediated ERK/CREB pathway, an important signaling cascade involved in memory formation.

Localized infusions of the partial alpha 7 nicotinic receptor agonist SSR180711 evoke rapid and transient increases in prefrontal glutamate release

The ability of local infusions of the alpha 7 nicotinic acetycholine receptor (α7 nAChR) partial agonist SSR180711 to evoke glutamate release in prefrontal cortex was determined in awake rats using a microelectrode array. Infusions of SSR180711 produced dose-dependent increases in glutamate levels. The lower dose (1.0μg in 0.4μL) evoked a rapid rise (∼1.0s) in glutamate (1.41±0.30μM above baseline). The higher dose (5.0μg) produced a similarly rapid, yet larger increase (3.51±0.36μM above baseline). After each dose, the glutamate signal was cleared to basal levels within 7–18s. SSR180711-evoked glutamate was mediated by the α7 nAChR as co-infusion of the selective α7 nAChR antagonist α-bungarotoxin (10.0μM)+SSR1808711 (5.0μg) reduced the effect of 5.0μg alone by 87% (2.62 vs. 0.35μM). Finally, the clearance of the SSR180711 (5.0μg)-evoked glutamate was bidirectionally affected by drugs that inhibited (threo-beta-benzyl-oxy-aspartate (TβOA), 100.0μM) or facilitated (ceftriaxalone, 200mg/kg, i.p.) excitatory amino acid transporters. TβOA slowed both the clearance (s) and rate of clearance (μM/s) by 10-fold, particularly at the mid-late stages of the return to baseline. Ceftriaxone reduced the magnitude of the SSR180711-evoked increase by 65%. These results demonstrate that pharmacological stimulation of α7 nAChRs within the prefrontal cortex is sufficient to evoke rapid yet transient increases in glutamate levels. Such increases may underlie the cognition-enhancing effects of the drug in animals; further justifying studies on the use of α7 nAChR-positive modulators in treating cognition-impairing disorders in humans.

P.1.j.014 Effects of chronic etanercept, a TNF-α inhibitor, on cognitive deficits in diabetic rats

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as therapeutic approaches for managing cognitive deficits in Alzheimer’s disease (AD). Present study was designed to evaluate the effect of α7 nAChR selective activation by PHA-543613 (PHA) on beta-amyloid (Aβ)25–35-mediated cognitive deficits in mice.For this purpose, PHA (1 mg/kg, i.p.), a selective α7 nAChR agonist, and galantamine (Gal) (3 mg/kg, s.c.), an acetylcholine-esterase inhibitor (AChEI) effects on α7 nAChR were tested in Aβ25–35-received (intracerebroventricular, 10 nmol) mice model of AD. Methyllycaconitine (MLA) (1 mg/kg, i.p.), a α7 nAChR antagonist, was used for receptor blockage effects evaluation. Working and reference memory in animals was assessed by the Morris water maze (MWM) task. The mRNA and protein levels of α7 subunit were analyzed by real-time PCR and Western blotting, respectively.PHA and Gal, ameliorate Aβ-impaired working and reference memory. However, Gal had less effect than PHA in this regard. Pretreatment with MLA reverses both Gal and PHA effects in MWM. PHA and Gal treatment prevent Aβ-induced α7 subunit protein reduction, but Gal has lesser effect than PHA. This effect blocked by pretreatment with MLA. In neither the pretreatment nor treatment group, the mRNA levels of nAChR α7 subunit were significantly changed.Therefore, α7 nAChR activation, reduces Aβ-induced cognitive deficits and increases the α7 protein level and subsequent neuron survival. However, blockage of receptor, increases Aβ toxicity and cognitive impairment and reduces the α7 nAChR protein level and flowing neuroprotection.

Volatile anesthetic isoflurane inhibits LTP induction of hippocampal CA1 neurons through α4β2 nAChR subtype-mediated mechanisms

Background and purposeVolatile anesthetic isoflurane contributes to postoperative cognitive dysfunction and inhibition of long-term potentiation (LTP), a synaptic model of learning and memory, but the mechanisms are uncertain. Central neuronal α4β2 subtype nicotinic acetylcholine receptors (nAChRs) are involved in the induction of LTP in the hippocampus. Isoflurane inhibits α4β2 nAChRs at concentrations lower than those used for anesthesia. Therefore, we hypothesized that isoflurane-inhibited LTP induction of hippocampal CA1 neurons via α4β2 nAChRs subtype inhibition. MethodsTransverse hippocampal slices (400μm thick) were obtained from male rats (6–8 weeks old). Population spikes were evoked using extracellular electrodes by electrical stimulation of the Schaffer collateral-commissural pathway of rat hippocampal slices. LTP was induced using high frequency stimulation (HFS; 100Hz, 1s). Clinically relevant concentrations (0.125-0.5mM) of isoflurane with or without nicotine (nAChRs agonist), mecamylamine (nAChRs antagonist), 3-[2(S)-2-azetidinylmethoxy] pyridine (A85380) and epibatidine (α4β2 nAChRs agonist), dihydro β erythroidine (DHβE) (α4β2 nAChRs antagonist) were added to the perfusion solution 20min before HFS to test their effects on LTP by HFS respectively. ResultsA brief HFS induced stable LTP in rat hippocampal slices, but LTP was significantly inhibited in the presence of isoflurane at concentrations of 0.125-0.5mM. The inhibitive effect of isoflurane on LTP was not only reversible and could be prevented by nAChRs agonist nicotine and α4β2 nAChRs agonist A85380 and epibatidine, but also mimicked and potentiated by nAChRs antagonist mecamylamine and α4β2 nAChRs antagonist DHβE. ConclusionsInhibition of α4β2 nAChRs subtype of hippocampus participates in isoflurane-mediated LTP inhibition.

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3β4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca2+ flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.

Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor: ANTAGONIST BINDING SITE AND MODE OF RECEPTOR INHIBITION REVEALED

The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4β2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(β2)2 nAChRs.

Contribution of CA3 and CA1 pyramidal neurons to the tonic α7 nAChR-dependent glutamatergic input to CA1 pyramidal neurons

The Schaffer collaterals are among the major glutamatergic inputs to CA1 pyramidal neurons, the primary output of the hippocampus, which also receive sparse recurrent inputs from pyramidal neurons in the CA1 field. Although tonically active α7 nicotinic acetylcholine receptors (nAChRs) have been shown to sustain spontaneous glutamate transmission to CA1 pyramidal neurons in hippocampal slices under resting conditions, it remains to be determined whether these receptors are those expressed by CA3 or CA1 pyramidal neurons. This study was designed to test the hypothesis that the CA3 field of the hippocampus is a significant source of α7 nAChR-sustained glutamatergic transmission to CA1 pyramidal neurons. To this end, spontaneous excitatory postsynaptic currents (EPSCs) were recorded from CA1 and CA3 pyramidal neurons in intact rat hippocampal slices as well as from CA1 pyramidal neurons in CA3-ablated slices under various experimental conditions. Surgical removal of the CA3 region from the slices reduced by 20% the frequency of spontaneous EPSCs recorded from CA1 pyramidal neurons. This finding is in agreement with the concept that the CA3 field contributes significantly to the maintenance of spontaneous glutamatergic synaptic activity in CA1 pyramidal neurons. In addition, the α7 nAChR antagonist methyllycaconitine (MLA, 10nM) reduced the frequency of spontaneous EPSCs recorded from CA1 pyramidal neurons by 30% in intact slices and 12% in CA3-ablated slices. Taken together, these results demonstrate that tonically active α7 nAChRs in CA3 pyramidal neurons and/or in the Mossy fibers that innervate the CA3 pyramidal neurons do in fact contribute to the maintenance of glutamatergic synaptic activity in CA1 pyramidal neurons of hippocampal slices under resting conditions.

Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: A model of cholinergic capacity limits

Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/− mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/− mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/− mice. Additionally, CHT+/− mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/− mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/− cholinergic terminals. Further analyses of molecular and functional compensations in the CHT+/− model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders.

Novel 2-(substituted benzyl)quinuclidines inhibit human α7 and α4β2 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 α4β2 nicotinic receptors (nAChRs). The antagonistic potency of these compounds was tested by Ca2+ influx assays on cells expressing the hα7 or hα4β2 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 hα4β2 nAChR ion channel domain. These results indicate that the methiodides are competitive antagonists of the hα7 nAChR but noncompetitive antagonists of the hα4β2 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 hα4β2 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.

Isolation and characterization of α-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors

A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3β2, α3α5β2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3β2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3β2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.