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.     

Dual effect of lobeline on α4β2 rat neuronal nicotinic receptors

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy, and the overexpression of P-glycoprotein (P-gp) is one of the major mechanisms of MDR. In this paper, we evaluated the effects of two new milbemycin compounds, milbemycin β(14) and secomilbemycin D, isolated from fermentation broth of S. bingchenggensis on reversing MDR of adriamycin-resistant human breast carcinoma (MCF-7/adr) cells. We observed that the both milbemycins (5 μM) showed strong potency to increase adriamycin cytotoxicity toward MCF-7/adr cells with reversal fold (RF) of 13.5 and 10.59, respectively. In addition, the mechanisms of milbemycins on reversing P-gp-mediated MDR demonstrated that they significantly increased the accumulations of adriamycin and Rh123 via inhibiting P-gp efflux in MCF-7/adr cells. Furthermore, the results also revealed that milbemycin β(14) and secomilbemycin D could regulate down the expression of P-gp, but not affect the expression of MDR1 gene. In conclusion, our observations suggest that the two new milbemycin compounds probably represent the promising agents for reversing MDR in cancer therapy.     

Atypical antipsychotics as noncompetitive inhibitors of α4β2 and α7 neuronal nicotinic receptors

It has been suggested that the interaction of antipsychotic medications with neuronal nicotinic receptors may increase the cognitive dysfunction associated with schizophrenia and may explain why current therapies only partially address this core feature of the illness. In the present studies we compared the effects of the atypical antipsychotics quetiapine, clozapine and N-desmethylclozapine to those of the typical antipsychotics haloperidol and chlorpromazine on the α4β2 and α7 nicotinic receptor subtypes. The binding of [³H]-nicotine to rat cortical α4β2 receptors and [³H]-methyllycaconitine to rat hippocampal α7 receptors was not affected by any of the compounds tested. However, Rb⁺ efflux evoked either by nicotine or the selective α4β2 agonist TC-1827 from α4β2 receptors expressed in SH-EP1 cells and nicotine-evoked [³H]-dopamine release from rat striatal synaptosomes were non-competitively inhibited by all of the antipsychotics. Similarly, α-bungarotoxin-sensitive epibatidine-evoked [³H]-norepinephrine release from rat hippocampal slices and acetylcholine-activated currents of α7 nicotinic receptors expressed in oocytes were inhibited by haloperidol, chlorpromazine, clozapine and N-desmethylclozapine. The inhibitory effects on nicotinic receptor function produced by the antipsychotics tested occurred at concentrations similar to plasma levels achieved in schizophrenia patients, suggesting that they may lead to clinically relevant effects on cognition.     

The additional ACh binding site at the α4(+)/α4(?) interface of the (α4β2)2α4 nicotinic ACh receptor contributes to desensitization

BACKGROUND AND PURPOSE

Nicotinic ACh (α4β2)₂α4 receptors are highly prone to desensitization by prolonged exposure to low concentrations of agonist. Here, we report on the sensitivity of the three agonist sites of the (α4β2)₂α4 to desensitization induced by prolonged exposure to ACh. We present electrophysiological data that show that the agonist sites of the (α4β2)₂α4 receptor have different sensitivity to desensitization and that full receptor occupation decreases sensitivity to desensitization.

EXPERIMENTAL APPROACH

Two-electrode voltage-clamp electrophysiology was used to study the desensitization of concatenated (α4β2)₂α4 receptors expressed heterologously in Xenopus oocytes. Desensitization was assessed by measuring the degree of functional inhibition caused by prolonged exposure to ACh, as measured under equilibrium conditions. We used the single-point mutation α4W182A to measure the contribution of individual agonist sites to desensitization.

KEY RESULTS

(α4β2)₂α4 receptors are less sensitive to activation and desensitization by ACh than (α4β2)₂β2 receptors. Incorporation of α4W182A into any of the agonist sites of concatenated (α4β2)₂α4 receptors decreased sensitivity to activation and desensitization but the effects were more pronounced when the mutation was introduced into the α4(+)/α4(−) interface.

CONCLUSIONS AND IMPLICATIONS

The findings suggest that the agonist sites in (α4β2)₂α4 receptors are not functionally equivalent. The agonist site at the α4(+)/α4(−) interface defines the sensitivity of (α4β2)₂α4 receptors to agonist-induced activation and desensitization. Functional differences between (α4β2)₂α4 and (α4β2)₂β2 receptors might shape the physiological and behavioural responses to nicotinic ligands when the receptors are exposed to nicotinic ligands for prolonged periods of times.     

Interaction of bispyridinium compounds with the orthosteric binding site of human α7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs)

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human α7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K_i in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC₅₀ > 400 μM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds.     

A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

AIM: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators. METHODS: Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. RESULTS: IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. CONCLUSIONS: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.     

Ligands selective for α4β2 but not α3β4 or α7 nicotinic receptors generalise to the nicotine discriminative stimulus in the rat

RATIONALE: Nicotine produces behavioural effects that are potentially related to its interaction with diverse nicotinic acetylcholine receptor populations. Evidence from gene deletion studies suggests that the interoceptive stimulus properties of nicotine are mediated by heteromeric high-affinity receptors containing alpha4beta2 subunits. Mice lacking beta2 subunits do not discriminate nicotine (Shoaib et al., Neuropharmacology, 42:530-539, 2002), and nicotine does not elicit dopamine release in these animals (Grady et al., J Neurochem, 76:258-268, 2001). The stimulus properties of nicotine can be detected in rats using a two-lever operant drug discrimination paradigm, allowing them to be classified pharmacologically using ligands with selectivity for receptors containing alpha4beta2, alpha3beta4 or alpha7 subunits. MATERIALS AND METHODS: Rats trained to discriminate 0.4 mg/kg nicotine from vehicle were given the nicotinic receptor agonists, cytisine, varenicline, TC2559, ABT-594, A-85380 (all having high affinity but varying selectivity for alpha4beta2-containing receptors), and WO 03/062224 and WO 01/60821A1 (selective for beta4- and alpha7-containing receptors, respectively). In separate studies, WO 03/062224 was used as the training stimulus. RESULTS: Nicotine, TC-2559, A-85380 and ABT-594 showed dose-dependent and complete stimulus substitution, whilst WO 03/062224 and WO 01/60821A1 were completely without effect. Cytisine and varenicline showed partial generalisation, consistent with their partial agonist activity at nicotinic receptors eliciting dopamine release in rat striatal slices. After almost 50 training sessions with WO 03/062224, there was no clear evidence that an alpha3beta4 receptor agonist could sustain a discriminable stimulus. CONCLUSION: Substitution to the nicotine discriminative stimulus required high-affinity and high intrinsic activity at beta2 but not at beta4- or at alpha7-containing nicotinic receptors.     

Deconstruction of the α4β2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine

Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.     

Chronic inhibition of α4β2 nicotinic receptors in the ventral hippocampus of rats: impacts on memory and nicotine response

RATIONALE: Acute and chronic systemic nicotine administration has been shown to cause significant spatial memory improvement. The critical nicotinic receptor subtypes for this effect and their location are still being determined. Nicotinic receptors in the ventral hippocampus have been found to be critically involved in memory. Acute ventral hippocampal infusions of dihydro-beta-erythroidine (DHbetaE), an alpha4beta2 nicotinic receptor antagonist, impaired spatial memory of rats in the radial-arm maze. OBJECTIVES: The current study used chronic ventral hippocampal infusion of DHbetaE as a model of nicotinic receptor loss such as that which occurs in Alzheimer's disease. The therapeutic effect of systemic nicotine treatment in reversing the DHbetaE-induced memory impairment was determined. METHODS: Rats were pretrained to asymptotic levels of performance on the eight-arm radial maze. Then, they were implanted with bilateral infusion cannulae in the ventral hippocampus, through which 0, 33.3, or 100 microg/side/day of DHbetaE was continuously infused for 4 weeks. The rats were retested on the eight-arm maze throughout infusion period and after withdrawal, and the interaction of acute systemic nicotine injections on memory was tested. RESULTS: The higher (100 microg/side/day) but not the lower (33.3 microg/side/day) DHbetaE dose caused a significant spatial memory impairment. Acute systemic nicotine injections (0, 0.1, 0.2, and 0.4 mg/kg, subcutaneous) attenuated the memory impairing effects of 100 microg/side/day of DHbetaE. There was no significant effect on response latency with the chronic DHbetaE infusion. Acute systemic nicotine infusions did significantly speed responding, an effect which was reversed by chronic hippocampal infusions of DHbetaE. After withdrawal there were no significant lasting effects on choice accuracy or response latency. Wet-dog shakes were significantly elevated during chronic hippocampal DHbetaE administration with no effect during the withdrawal period. CONCLUSIONS: These results indicate that chronic inhibition of a subset of nicotinic receptors in the hippocampus results in a significant impairment in the spatial memory choice accuracy. The ability of nicotine to attenuate the impairment supports the development of nicotinic agonist therapy of syndromes, such as Alzheimer's disease, that involve a chronic decrease in the activity of the alpha4beta2 nicotinic receptors and memory impairment.     

PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization

Background and Purpose

Nicotinic (ACh) receptor recovery from desensitization is modulated by PKC, but the PKC isozymes and the phosphorylation sites involved have not been identified. We investigated whether PKCε phosphorylation of α₄β₂ nAChRs regulates receptor recovery from desensitization.

Experimental Approach

Receptor recovery from desensitization was investigated by electrophysiological characterization of human α₄β₂ nAChRs. Phosphorylation of the α₄ nAChR subunit was assessed by immunoblotting of mouse synaptosomes. Hypothermia induced by sazetidine-A and nicotine was measured in Prkce^−/− and wild-type mice.

Key Results

Inhibiting PKCε impaired the magnitude of α₄β₂ nAChR recovery from desensitization. We identified five putative PKCε phosphorylation sites in the large intracellular loop of the α₄ subunit, and mutating four sites to alanines also impaired recovery from desensitization. α₄ nAChR subunit phosphorylation was reduced in synaptosomes from Prkce^−/− mice. Sazetidine-A-induced hypothermia, which is mediated by α₄β₂ nAChR desensitization, was more severe and prolonged in Prkce^−/− than in wild-type mice.

Conclusions and Implications

PKCε phosphorylates the α₄ nAChR subunit and regulates recovery from receptor desensitization. This study illustrates the importance of phosphorylation in regulating α₄β₂ receptor function, and suggests that reducing phosphorylation prolongs receptor desensitization and decreases the number of receptors available for activation.Tables of Links

Structural differences determine the relative selectivity of nicotinic compounds for native α4β2*-, α6β2*-, α3β4*- and α7-nicotine acetylcholine receptors

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, α4β2* and α6β2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The α4β2*-nAChRs are widely distributed, while α6β2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. α4β2*-nAChR also modulate GABA release in these areas.Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing hα7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, α4β2*, α6β2*, α7 and α3β4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at α4β2* and α6β2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for α6β2*-nAChR.     

Brain α4β2 nicotinic acetylcholine receptors are involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats

Recently, we reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (a non-selective agonist of nicotinic acetylcholine receptors) elevates plasma noradrenaline and adrenaline through brain nicotinic acetylcholine receptor-mediated mechanisms in rats. In the present study, we characterized the receptors involved in these responses using selective agonists and antagonists of nicotinic acetylcholine receptor subtypes in anesthetized rats. (±)-Epibatidine (5 and 10 nmol/animal, i.c.v.) and (−)-nicotine (250 and 500 nmol/animal, i.c.v.) both elevated plasma noradrenaline and adrenaline (adrenaline > noradrenaline) but the former was more efficient than the latter. The (±)-epibatidine (5 nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by dihydro-β-erythroidine (a selective antagonist of α4β2 nicotinic acetylcholine receptors) (100 and 300 nmol/animal, i.c.v.), while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors) (100 and 300 nmol/animal, i.c.v.) had no effect on the (±)-epibatidine-induced responses. RJR-2403 (a selective agonist of α4β2 nicotinic acetylcholine receptors) (2.5 and 5 μmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline (adrenaline > noradrenaline), while PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors) (2.5 and 5 μmol/animal, i.c.v.) had no effect. Furthermore, the RJR-2403 (5 μmol/animal, i.c.v.)-induced responses were abolished by acute bilateral adrenalectomy. Immunohistochemical procedures demonstrated the expression of α4 and β2 nicotinic acetylcholine receptor subunits on the spinally projecting hypothalamic paraventricular neurons. Taken together, brain α4β2 nicotinic acetylcholine receptors seem to be involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats.     

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.     

Progress and challenges in the study of α6-containing nicotinic acetylcholine receptors

Recent progress has been made in the understanding of the anatomical distribution, composition, and physiological role of nicotinic acetylcholine receptors containing the α6 subunit. Extensive study by many researchers has indicated that a collection of α6-containing receptors representing a nicotinic sub-family is relevant in preclinical models of nicotine self-administration and locomotor activity. Due to a number of technical difficulties, the state of the art of in vitro model systems expressing α6-containing receptors has lagged behind the state of knowledge of native α6 nAChR subunit composition. Several techniques, such as the expression of chimeric and concatameric α6 subunit constructs in oocytes and mammalian cell lines have been employed to overcome these obstacles. There remains a need for other critical tools, such as selective small molecules and radioligands, to advance the field of research and to allow the discovery and development of potential therapeutics targeting α6-containing receptors for smoking cessation, Parkinson's disease and other disorders.     

Nicotine-induced enhancement of responding for conditioned reinforcement in rats: Role of prior nicotine exposure and α4β2 nicotinic receptors

Rationale

Stimuli associated with nicotine can become motivationally significant and may play a role in tobacco dependence. Previous work indicates that nicotine enhances responding for a conditioned reinforcer (CR).

Objectives

These studies examined the effects of prior exposure to nicotine on responding for a CR, persistence of this response, and the role of α4β2 or α7 nicotinic receptor subtypes.

Methods

Water deprived rats were given 13 Pavlovian conditioning sessions where a light/tone conditioned stimulus (CS) was paired with the delivery of water. Then, rats were presented with two levers: one delivered the CS (now a CR), the other was inactive. Experiments examined the effect of nicotine administered prior to Pavlovian conditioning sessions on approach behavior during CS presentations, operant responding for CR in the presence and absence of nicotine, and the persistence of responding for CR. The effects of nicotinic acetylcholine receptor (nAChR) antagonism with mecamylamine and α4β2 or α7 nAChR antagonism with dihydro-beta-erythroidine (DHβE) or methyllycaconitine (MLA) on nicotine-enhanced responding for CR were examined.

Results

Nicotine enhanced approach behavior during CS presentations and potentiated operant responding for CR, an effect sensitized as a result of nicotine exposure during conditioning. Responding for CR and its potentiation by nicotine was stable over multiple tests. Enhanced responding for the CR induced by nicotine was blocked by mecamylamine and DHβE, but not MLA.

Conclusions

Nicotine enhances Pavlovian discriminated approach and shows sensitized nicotine-induced enhancements in responding for CR, an effect depending on α4β2 nAChRs.