Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are ligand-gated ion conducting transmembrane channels from the Cys-loop receptor super-family. The α4β2 subtype is the predominant heteromeric subtype of nicotinic receptors found in the brain. Allosteric modulators for α4β2 receptors interact at a site other than the orthosteric site where acetylcholine binds. Many compounds which act as allosteric modulators of the α4β2 receptors have been identified, with both positive and negative effects. Such allosteric modulators either increase or decrease the response induced by agonist on the α4β2 receptors. Here we discuss the concept of allosterism as it pertains to the α4β2 receptors and summarize the important features of allosteric modulators for this nicotinic receptor subtype.     

Quercetin Inhibits ��3��4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT_3A and α7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal α3β4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal α3 and β4 subunits. Treatment with acetylcholine elicited an inward peak current (I_ACh) in oocytes expressing α3β4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited I_ACh in oocytes expressing α3β4 nicotinic acetylcholine receptors. The inhibition of I_ACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC₅₀) of quercetin was 14.9±0.8 µM in oocytes expressing α3β4 nicotinic acetylcholine receptor. The inhibition of I_ACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate α3β4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.     

Nicotinic acetylcholine receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse     

α7 Nicotinic acetylcholine receptors in the central amygdaloid nucleus alter naloxone-induced withdrawal following a single exposure to morphine

Rationale  

Negative motivational withdrawal from acute opiate dependence was induced by an opioid antagonist, and the withdrawal signs prevented by pretreatment with nicotine.     

N-n-alkylnicotinium analogs, a novel class of antagonists at α4β2* Nicotinic acetylcholine receptors: Inhibition of S(-)-nicotine-evoked 86Rb+Efflux from rat thalamic synaptosomes

Pyridine N-n-alkylation of S(-)-nicotine (NIC) affords N-n-alkylnicotinium analogs, previously shown to competitively inhibit [(3)H]NIC binding and interact with alpha4beta2* nicotinic receptors (nAChRs). The present study determined the ability of the analogs to inhibit NIC-evoked (86)Rb(+) efflux from rat thalamic synaptosomes to assess functional interaction with alpha4beta2* nAChRs. In a concentration-dependent manner, NIC evoked (86)Rb(+) efflux (EC(50) = 170 nmol/L). Analog-induced inhibition of NIC-evoked (86)Rb(+) efflux varied over a approximately 450-fold range. Analogs with long n-alkyl chain lengths (C(9)-C(12)) inhibited efflux in the low nmol/L range (IC(50) = 9-20 nmol/L), similar to dihydro-beta-erythroidine (IC(50) = 19 nmol/L). Compounds with shorter n-alkyl chain lengths (C(1)-C(8)) produced inhibition in the low micromol/L range (IC(50) = 3-12 micromol/L). C(10) and C(12) analogs completely inhibited NIC-evoked efflux, whereas C(1-9) analogs produced maximal inhibition of only 10% to 60%. While the C(10) analog N-n-decylnicotinium iodide (NDNI) did not produce significant inhibition of NIC-evoked dopamine release in previously reported studies, NDNI possesses high affinity for [(3)H]NIC binding sites (K(i) = 90 nmol/L) and is a potent and efficacious inhibitor of NIC-evoked (86)Rb(+) efflux as demonstrated in the current studies. Thus, NDNI is a competitive, selective antagonist at alpha4beta2* nAChRs.     

Partial nicotinic acetylcholine (��4��2) agonists as promising new medications for smoking cessation

Objective:

To review the pharmacology, clinical efficacy and safety of partial agonists of α4β2 nicotinic acetylcholine receptor.

Data Sources:

Primary literature and review articles were obtained via a PUBMED search (1988-August 2006) using the key terms smoking cessation, partial agonist alpha4beta2 nicotinic acetylcholine receptor, varenicline, cytisine and SSR591813. Additional studies and abstracts were identified from the bibliographies of reviewed literature.

Study Selection and Data Extraction:

Studies and review articles related to varenicline, cytisine and the partial agonist alpha4beta2 nicotinic acetylcholine receptor were reviewed.

Data Synthesis:

Smoking is widely recognized as a serious health problem. Smoking cessation has major health benefits. According to the US Public Health Services, all patients attempting to quit smoking should be encouraged to use one or more effective pharmacotherapy. Currently, along with nicotine replacement therapy, bupropion, nortriptyline and clonidine, are the mainstay of pharmacotherapy. More than ¾ of patients receiving treatment for smoking cessation return to smoking within the first year. Nicotine, through stimulating α4β2 nAChR, releases dopamine in the reward pathway. Partial agonist of α4β2 nAChR elicits moderate and sustained release of dopamine, which is countered during the cessation attempts; it simultaneously blocks the effects of nicotine by binding with α4β2 receptors during smoking. Recently, varenicline, a partial agonist at α4β2 nAChR, has been approved by the FDA (Food and Drug Administration) for smoking cessation.

Conclusion:

Partial agonist α4β2 nAChR appears to be a promising target in smoking cessation. Varenicline of this group is approved for treatment of smoking cessation by the FDA in May 2006.     

Role of β2-containing nicotinic acetylcholine receptors in auditory event-related potentials

Rationale  Nicotine improves sensory processing in schizophrenic individuals, as measured by changes in auditory event-related potentials (ERPs). Nicotine administration also alters ERPs in mice by increasing the amplitude and gating of the P20 ERP component while decreasing the amplitude of the N40 ERP component. Less is known about the role of specific nicotinic acetylcholine receptor (nAChR) subtypes. Objectives  In this study, we examined whether nAChRs containing the β2 subunit contribute to nicotine’s effects on auditory ERPs. Materials and methods  We tested the effect of nicotine in wild-type mice and mice lacking the β2 nAChR subunit. Mice underwent stereotaxic implantation of stainless steel electrodes located in the CA3 region of the hippocampus, and 50 paired click stimuli were delivered during each drug condition. Results  There was no significant difference in P20 or N40 amplitude or gating between genotypes during the control condition, suggesting that β2-containing receptors are not essential for the baseline auditory ERP response. Nicotine increased P20 amplitude and enhanced gating in wild-type and β2 knockout mice, but only decreased N40 amplitude in wild-type mice. There was no effect of nicotine on N40 gating in either genotype. Conclusions  β2-containing receptors are necessary for nicotine’s effects on the N40 component of the mouse auditory ERP. These results suggest that β2-containing nAChRs modulate sensory processing and may serve as a therapeutic target in schizophrenic individuals.     

α6β2* and α4β2* nicotinic acetylcholine receptors as drug targets for Parkinson's disease

Parkinson's disease is a debilitating movement disorder characterized by a generalized dysfunction of the nervous system, with a particularly prominent decline in the nigrostriatal dopaminergic pathway. Although there is currently no cure, drugs targeting the dopaminergic system provide major symptomatic relief. As well, agents directed to other neurotransmitter systems are of therapeutic benefit. Such drugs may act by directly improving functional deficits in these other systems, or they may restore aberrant motor activity that arises as a result of a dopaminergic imbalance. Recent research attention has focused on a role for drugs targeting the nicotinic cholinergic systems. The rationale for such work stems from basic research findings that there is an extensive overlap in the organization and function of the nicotinic cholinergic and dopaminergic systems in the basal ganglia. In addition, nicotinic acetylcholine receptor (nAChR) drugs could have clinical potential for Parkinson's disease. Evidence for this proposition stems from studies with experimental animal models showing that nicotine protects against neurotoxin-induced nigrostriatal damage and improves motor complications associated with l-DOPA, the "gold standard" for Parkinson's disease treatment. Nicotine interacts with multiple central nervous system receptors to generate therapeutic responses but also produces side effects. It is important therefore to identify the nAChR subtypes most beneficial for treating Parkinson's disease. Here we review nAChRs with particular emphasis on the subtypes that contribute to basal ganglia function. Accumulating evidence suggests that drugs targeting α6β2* and α4β2* nAChR may prove useful in the management of Parkinson's disease.     

Effects of chronic sazetidine-A, a selective α4β2 neuronal nicotinic acetylcholine receptors desensitizing agent on pharmacologically-induced impaired attention in rats

Rationale

Nicotine and nicotinic agonists have been shown to improve attentional function. Nicotinic receptors are easily desensitized, and all nicotinic agonists are also desensitizing agents. Although both receptor activation and desensitization are components of the mechanism that mediates the overall effects of nicotinic agonists, it is not clear how each of the two opposed actions contributes to attentional improvements. Sazetidine-A has high binding affinity at α4β2 nicotinic receptors and causes a relatively brief activation followed by a long-lasting desensitization of the receptors. Acute administration of sazetidine-A has been shown to significantly improve attention by reversing impairments caused by the muscarinic cholinergic antagonist scopolamine and the NMDA glutamate antagonist dizocilpine.

Methods

In the current study, we tested the effects of chronic subcutaneous infusion of sazetidine-A (0, 2, or 6 mg/kg/day) on attention in Sprague-Dawley rats. Furthermore, we investigated the effects of chronic sazetidine-A treatment on attentional impairment induced by an acute administration of 0.02 mg/kg scopolamine.

Results

During the first week period, the 6-mg/kg/day sazetidine-A dose significantly reversed the attentional impairment induced by scopolamine. During weeks 3 and 4, the scopolamine-induced impairment was no longer seen, but sazetidine-A (6 mg/kg/day) significantly improved attentional performance on its own. Chronic sazetidine-A also reduced response latency and response omissions.

Conclusions

This study demonstrated that similar to its acute effects, chronic infusions of sazetidine-A improve attentional performance. The results indicate that the desensitization of α4β2 nicotinic receptors with some activation of these receptors may play an important role in improving effects of sazetidine-A on attention.     

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

Rationale  

Nicotine, a major addictive component of tobacco, has been suggested to provoke impulsivity by activating central α4β2 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.     

EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors

EVP-6124, (R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was evaluated here in vitro and in vivo. In binding and functional experiments, EVP-6124 showed selectivity for α7 nAChRs and did not activate or inhibit heteromeric α4β2 nAChRs. EVP-6124 had good brain penetration and an adequate exposure time. EVP-6124 (0.3 mg/kg, p.o.) significantly restored memory function in scopolamine-treated rats (0.1 mg/kg, i.p.) in an object recognition task (ORT). Although donepezil at 0.1 mg/kg, p.o. or EVP-6124 at 0.03 mg/kg, p.o. did not improve memory in this task, co-administration of these sub-efficacious doses fully restored memory. In a natural forgetting test, an ORT with a 24 h retention time, EVP-6124 improved memory at 0.3 mg/kg, p.o. This improvement was blocked by the selective α7 nAChR antagonist methyllycaconitine (0.3 mg/kg, i.p. or 10 μg, i.c.v.). In co-application experiments of EVP-6124 with acetylcholine, sustained exposure to EVP-6124 in functional investigations in oocytes caused desensitization at concentrations greater than 3 nM, while lower concentrations (0.3-1 nM) caused an increase in the acetylcholine-evoked response. These actions were interpreted as representing a co-agonist activity of EVP-6124 with acetylcholine on α7 nAChRs. The concentrations of EVP-6124 that resulted in physiological potentiation were consistent with the free drug concentrations in brain that improved memory performance in the ORT. These data suggest that the selective partial agonist EVP-6124 improves memory performance by potentiating the acetylcholine response of α7 nAChRs and support new therapeutic strategies for the treatment of cognitive impairment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Partial Agonists of the ��3��4* Neuronal Nicotinic Acetylcholine Receptor Reduce Ethanol Consumption and Seeking in Rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2^* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.     

Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors

BACKGROUND AND PURPOSE

Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

EXPERIMENTAL APPROACH

Effects of NS9283 were evaluated in vitro using fluorescence-based Ca²⁺ imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats.

KEY RESULTS

NS9283 was shown to increase agonist-evoked response amplitude of (α4)₃(β2)₂ nACh receptors in electrophysiology paradigms. (α2)₃(β2)₂, (α2)₃(β4)₂ and (α4)₃(β4)₂ were modulated to comparable extents, but no effects were detected at α3-containing or any 2α : 3β stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHβE-sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP-disrupted pre-pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five-choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors.

CONCLUSIONS AND IMPLICATIONS

These results provide compelling evidence that positive allosteric modulators acting at the (α4)₃(β2)₂ nACh receptors can augment activity across a broad range of cognitive domains, and that α4β2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment.     

α7 nicotinic acetylcholine receptors in Alzheimer's disease: neuroprotective, neurotrophic or both?

One of the early signs of Alzheimer's disease is the impairment in hippocampus-based episodic memory function, which is improved through the enhancement of cholinergic transmission. Several studies suggest that α7 nicotinic receptor (nAChR) activation represents a useful therapeutic strategy for the cognitive impairments associated with early Alzheimer's disease as the α7 subtype of nicotinic acetylcholine receptors are expressed by basal forebrain cholinergic projection neurons as well as by their targets in the hippocampus. The current model for the cholinergic deficit in Alzheimer's disease posits that inappropriate accumulation of misfolded oligomeric aggregates of β-amyloid peptide leads to the dysfunction of the signaling mechanisms that support the cholinergic phenotype; this is manifested as an altered function of nicotinic acetylcholine receptors and the nerve-growth factor trophic support system that results in the loss of cholinergic markers and eventually cholinergic neurons from the basal forebrain cholinergic system. A view was confounded by the fact that α7 nAChRs and β-amyloid peptides have been shown to interact in vitro and in vivo, including human post-mortem AD brain. This review will begin with a brief overview of the basal forebrain cholinergic system, followed by a discussion of the current knowledge of the cholinergic deficit in Alzheimer's disease, then a summary of the cholinergic phenotype observed in transgenic Alzheimer's disease mouse models. We will also present our recent findings that support our hypothesis that the α7 nicotinic acetylcholine receptor performs both the neurotrophic and neuroprotective roles in the maintenance of the cholinergic phenotype and discusses potential mechanisms and implications for Alzheimer's disease therapy.     

α7β2 nicotinic acetylcholine receptors assemble, function, and are activated primarily via their α7-α7 interfaces

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. F?rster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.