Human Α4β2 nAChRs Research Articles

Insights into Crystallization of Neuronal Nicotinic α4β2 Receptor in Polarized Lipid Matrices

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this study, we performed crystallization screenings of purified human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2 nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC 16 complex gave a mobile fraction >0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases.

Delineating the activity of the potent nicotinic acetylcholine receptor agonists (+)-anatoxin-a and (-)-hosieine-A.

The affinity and thermodynamic parameters for the interactions of two naturally occurring neurotoxins, (+)-anatoxin-a and (-)-hosieine-A, with acetylcholine-binding protein were investigated using a fluorescence-quenching assay and isothermal titration calorimetry. The crystal structures of their complexes with acetylcholine-binding protein from Aplysia californica (AcAChBP) were determined and reveal details of molecular recognition in the orthosteric binding site. Comparisons treating AcAChBP as a surrogate for human α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the molecular features involved in ligand recognition and affinity for the protein targets are conserved. The ligands exploit interactions with similar residues as the archetypal nAChR agonist nicotine, but with greater affinity. (-)-Hosieine-A in particular has a high affinity for AcAChBP driven by a favorable entropic contribution to binding. The ligand affinities help to rationalize the potent biological activity of these alkaloids. The structural data, together with comparisons with related molecules, suggest that there may be opportunities to extend the hosieine-A scaffold to incorporate new interactions with the complementary side of the orthosteric binding site. Such a strategy may guide the design of new entities to target human α4β2 nAChR that may have therapeutic benefit.

Stoichiometry-Selective Antagonism of α4β2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics.

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4β2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4β2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)2(β2)3 and (α4)3(β2)2, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4β2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4β2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)2(β2)3 nAChRs than of (α4)3(β2)2 nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)2(β2)3 nAChRs with an IC50 of 26.4 ± 3.4 μM but displayed no significant inhibition of (α4)3(β2)2 nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4β2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the β2(+)/β2(−) subunit interface, which is consistent with its selective inhibition of (α4)2(β2)3. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties.

Levo-tetrahydropalmatine inhibits α4β2 nicotinic receptor response to nicotine in cultured SH-EP1 cells.

Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4β2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 μM) induced inward currents, co-application of l-THP (3 μM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 μM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4β2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4β2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42β23-nAChR) and cytisine-activated, low-sensitive one (α43β22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4β2-nAChR function, which may underlie its inhibition on nicotine addiction.

Identification of α4β2 nAChR interaction site with Aβ42 and development of tetrapeptide capable of breaking this interaction

AbstractBackgroundCholinergic dysfunction in Alzheimer’s disease may arise from selective death of cholinergic neurons caused by the interaction of Aβ peptide with nicotinic acetylcholine receptors (nAChRs). Thereby, compounds that prevent the interaction of Aβ with nAChRs are needed to address the cholinergic deficit. Here, we identify the interaction site between Aβ42 and one of the two most abundant nicotinic receptors ‐ α4β2 nAChR. Further, we propose a tetrapeptide that prevents this interaction and serves as a potential therapeutic to counteract the cholinergic deficit in AD.MethodSequence analysis was used to identify the binding site of Aβ42 to α4β2 nAChR (35HAEE38). Surface plasmon resonance was utilized to evaluate the interaction between HAEE peptide and Aβ16. A complex of human α4β2 nAChR with Aβ42 was modeled with bioinformatics approaches. Human α4β2 nAChR was expressed in X. laevis oocytes. The receptor currents in the presence of Aβ42 and HAEE were studied with a two‐electrode voltage clamp.ResultAnalysis of α4β2 nAChR sequence showed that N‐terminal fragment 35HAEE38 of the α4 subunit is ionic complementary to 11EVHH14 in Aβ, suggesting it to be a potential site of the interaction. Synthetic HAEE peptide demonstrated binding to Aβ16, and molecular modeling confirmed that interaction of Aβ42 with α4β2 nAChR can occur via 11EVHH14:35HAEE38 interface. Pre‐incubation with Aβ42 reduced the amplitude of ACh‐evoked response in α4β2 nAChR‐transfected X. laevis oocytes by 20% and increased the leakage current in oocytes. Co‐incubation of Aβ42 with HAEE tetrapeptide reduced the degree of inhibition. Washout of Aβ42 with HAEE‐containing solution restored the current amplitude and reduced the leakage current.Conclusion35HAEE38 is a potential binding site for Aβ42 on α4β2 nAChR. HAEE tetrapeptide corresponding to this site restores inhibition of α4β2 nAChR by Aβ42 opening a potential therapeutic avenue for cholinergic dysfunction in AD.Supported by RSF grant #19‐74‐30007.

Menthol Binding to the Human α4β2 Nicotinic Acetylcholine Receptor Facilitated by Its Strong Partitioning in the Membrane.

We utilize various computational methodologies to study menthol's interaction with multiple organic phases, a lipid bilayer, and the human α4β2 nicotinic acetylcholine receptor (nAChR), the most abundant nAChR in the brain. First, force field parameters developed for menthol are validated in alchemical free energy perturbation simulations to calculate solvation free energies of menthol in water, dodecane, and octanol and compare the results against experimental data. Next, umbrella sampling is used to construct the free energy profile of menthol permeation across a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The results from a flooding simulation designed to study the water-membrane partitioning of menthol in a POPC lipid bilayer are used to determine the penetration depth and the preferred orientation of menthol in the bilayer. Finally, employing both docking and flooding simulations, menthol is shown to bind to different sites on the human α4β2 nAChR. The most likely binding mode of menthol to a desensitized membrane-embedded α4β2 nAChR is identified to be via a membrane-mediated pathway in which menthol binds to the sites at the lipid-protein interface after partitioning in the membrane. A rare but distinct binding mode in which menthol binds to the extracellular opening of receptor's ion permeation pore is also reported.

Fulditoxin, representing a new class of dimeric snake toxins, defines novel pharmacology at nicotinic ACh receptors

Animal toxins have contributed significantly to our understanding of the neurobiology of receptors and ion channels. We studied the venom of the coral snake Micrurus fulvius fulvius and identified and characterized the structure and pharmacology of a new homodimeric neurotoxin, fulditoxin, that exhibited novel pharmacology at nicotinic ACh receptors (nAChRs). Fulditoxin was isolated by chromatography, chemically synthesized, its structure determined by X-ray crystallography, and its pharmacological actions on nAChRs characterized by organ bath assays and two-electrode voltage clamp electrophysiology. Fulditoxin's distinct 1.95-Å quaternary structure revealed two short-chain three-finger α-neurotoxins (α-3FNTxs) non-covalently bound by hydrophobic interactions and an ability to bind metal and form tetrameric complexes, not reported previously for three-finger proteins. Although fulditoxin lacked all conserved amino acids canonically important for inhibiting nAChRs, it produced postsynaptic neuromuscular blockade of chick muscle at nanomolar concentrations, comparable to the prototypical α-bungarotoxin. This neuromuscular blockade was completely reversible, which is unusual for snake α-3FNTxs. Fulditoxin, therefore, interacts with nAChRs by utilizing a different pharmacophore. Unlike short-chain α-3FNTxs that bind only to muscle nAChRs, fulditoxin utilizes dimerization to expand its pharmacological targets to include human neuronal α4β2, α7, and α3β2 nAChRs which it blocked with IC50 values of 1.8, 7, and 12 μM respectively. Based on its distinct quaternary structure and unusual pharmacology, we named this new class of dimeric Micrurus neurotoxins represented by fulditoxin as Σ-neurotoxins, which offers greater insight into understanding the interactions between nAChRs and peptide antagonists.

An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4β2 and α7 nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4β2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4–7 Å are pharmacophoric elements necessary for molecular recognition by α4β2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids—nicotine, nornicotine, anabasine and R-anatabine—potently activated α4β2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4β2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4β2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4β2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4β2 and α7 nAChRs. It also revealed human α4β2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

Growth and neurite stimulating effects of the neonicotinoid pesticide clothianidin on human neuroblastoma SH-SY5Y cells

Neonicotinoids are one of most widely used pesticides targeting nicotinic acetylcholine receptors (nAChRs) of insects. Recent epidemiological evidence revealed increasing amounts of neonicotinoids detected in human samples, raising the critical question of whether neonicotinoids affect human health. We investigated the effects of a neonicotinoid pesticide clothianidin (CTD) on human neuroblastoma SH-SY5Y cells as in vitro models of human neuronal cells. Cellular and functional effects of micromolar doses of CTD were evaluated by changes in cell growth, intracellular signaling activities and gene expression profiles. We examined further the effects of CTD on neuronal differentiation by measuring neurite outgrowth. Exposure to CTD (1–100 μM) significantly increased the number of cells within 24 h of culture. The nAChRs antagonists, mecamylamine and SR16584, inhibited this effect, suggesting human α3β4 nAChRs could be targets of neonicotinoids. We observed a transient intracellular calcium influx and increased phosphorylation of extracellular signal-regulated kinase 1/2 shortly after exposure to CTD. Transcriptome analysis revealed that CTD down-regulated genes involved in neuronal function (e.g., formation of filopodia and calcium ion influx) and morphology (e.g., axon guidance signaling and cytoskeleton signaling); these changes were reflected by a finding of increased neurite length during neuronal differentiation. These findings provide novel insight into the potential risks of neonicotinoids to the human nervous system.

Identification of the Initial Steps in Signal Transduction in the α4β2 Nicotinic Receptor: Insights from Equilibrium and Nonequilibrium Simulations

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission in the nervous system. These receptors have emerged as therapeutic targets in drug discovery for treating several conditions, including Alzheimer's disease, pain, and nicotine addiction. In this in silico study, we use a combination of equilibrium and nonequilibrium molecular dynamics simulations to map dynamic and structural changes induced by nicotine in the human α4β2 nAChR. They reveal a striking pattern of communication between the extracellular binding pockets and the transmembrane domains (TMDs) and show the sequence of conformational changes associated with the initial steps in this process. We propose a general mechanism for signal transduction for Cys-loop receptors: the mechanistic steps for communication proceed firstly through loop C in the principal subunit, and are subsequently transmitted, gradually and cumulatively, to loop F of the complementary subunit, and then to the TMDs through the M2-M3 linker.

Drysdalin, an antagonist of nicotinic acetylcholine receptors highlights the importance of functional rather than structural conservation of amino acid residues.

Snake venom neurotoxins are potent antagonists of nicotinic acetylcholine receptors (nAChRs). Here, we describe a novel member of class 3c long‐chain neurotoxin drysdalin from the venom of Drysdalia coronoides. Drysdalin lacks three of the eight conserved classical functional residues critical for nAChRs interaction. Despite such a drastic alteration of the functional site, recombinant drysdalin showed irreversible postsynaptic neurotoxicity with nanomolar potency and selectively antagonizes the rodent muscle (α1)2β1δε, and human α7 and α9α10 nAChRs, but had no significant activity at the human α3β2, α3β4, α4β2, and α4β4 nAChRs. Substitution of Leu34 and Ala37 residues with the conserved Arg had minimal impact on the potency whereas conserved Phe replacement of residue Arg30 substantially reduced or abolished inhibitory activity. In contrast, truncation of the 24‐residue long C‐terminal tail leads to complete loss in (a) activity at α9α10 nAChR; and (b) irreversibility with reduced potency at the muscle and α7 nAChRs. Overall, the non‐conserved Arg30 residue together with the uniquely long C‐terminal tail contribute to the inhibitory activity of drysdalin at the nAChRs suggesting, at least for drysdalin, functional rather than sequence conservation plays a critical role in determining the activity of the toxin.

Dual Antidepressant Duloxetine Blocks Nicotinic Receptor Currents, Calcium Signals and Exocytosis in Chromaffin Cells Stimulated with Acetylcholine.

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α3, α5, and α7 nAChRs and in cell lines expressing human α7, α3β4, or α4β2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC50 of 0.86 µM. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca2+]c), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na+ or Ca2+ channel currents neither the high-K+-elicited [Ca2+]c transients and secretion. Of interest was that in cell lines expressing human α7, α3β4, and α4β2 nAChRs, duloxetine blocked nicotinic currents with IC50 values of 0.1, 0.56, and 0.85 µM, respectively. Thus, in blocking α7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine.

The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors.

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

Molecular basis of reactive oxygen species-induced inactivation of α4β2 nicotinic acetylcholine receptors

The α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are the most widespread heteromeric nAChR subtype in the brain, mediating fast synaptic transmission. Previous studies showed that α4β2 nAChRs could be inactivated by reactive oxygen species (ROS), but the underlying mechanism is still obscure. We found that H2O2 induced the rundown of ACh-evoked currents in human α4β2 nAChRs and the replacement of the conserved cysteine in the M1–M2 linker of either α4 Cys245 or β2 Cys237 with an alanine residue could prevent the current rundown. Structurally, α4 Cys245 and β2 Cys237 are hypothesized to be in close proximity when the receptor is activated. Western blotting results showed that α4 and β2 subunits were cross-linked when the agonist-bound receptor encountered H2O2, which could be prevented by the substitution of the conserved cysteine in the M1–M2 linker to an alanine. Thus, when agonist bound to the receptor, α4 Cys245 and β2 Cys237 came close to each other and ROS oxidized these conserved cysteines, leading subunits to be cross-linked and trapping α4β2 nAChRs into the inactivation state. In addition, we mimicked an experimental Parkinson's disease (PD) model in PC12 cells and found that ROS, generated by 6-hydroxydopamine (6-OHDA), could cause the current rundown in α4β2 nAChRs, which may play a role in PD.

Homology Model and Ligand Binding Interactions of the Extracellular Domain of the Human <i>α</i>4<i>β</i>2 Nicotinic Acetylcholine Receptor

Addiction to nicotine, and possibly other tobacco constituents, is a major factor that contributes to the difficulties smokers face when attempting to quit smoking. Amongst the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 subtype plays an important role in mediating the addiction process. The characterization of human α4β2-ligand binding interactions provides a molecular framework for understanding ligand-receptor interactions, rendering insights into mechanisms of nicotine addiction and may furnish a tool for efficiently identifying ligands that can bind the nicotine receptor. Therefore, we constructed a homology model of human α4β2 nAChR and performed molecular docking and molecular dynamics (MD) simulations to elucidate the potential human α4β2-ligand binding modes for eleven compounds known to bind to this receptor. Residues V96, L97 and F151 of the α4 subunit and L111, F119 and F121 of the β2 subunit were found to be involved in hydrophobic interactions while residues S153 and W154 of the α4 subunit were involved in the formation of hydrogen bonds between the receptor and respective ligands. The homology model and its eleven ligand-bound structures will be used to develop a virtual screening program for identifying tobacco constituents that are potentially addictive.

Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

Regulation of the “neuronal” nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

Anxiolytic- and antidepressant-like effects of the methadone metabolite 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP)

The enhancement of GABAergic and monoaminergic neurotransmission has been the mainstay of pharmacotherapy and the focus of drug-discovery for anxiety and depressive disorders for several decades. However, the significant limitations of drugs used for these disorders underscores the need for novel therapeutic targets. Neuronal nicotinic acetylcholine receptors (nAChRs) may represent one such target. For example, mecamylamine, a non-competitive antagonist of nAChRs, displays positive effects in preclinical tests for anxiolytic and antidepressant activity in rodents. In addition, nicotine elicits similar effects in rodent models, possibly by receptor desensitization. Previous studies (Xiao et al., 2001) have identified two metabolites of methadone, EMDP (2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline) and EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), which are considered to be inactive at opiate receptors, as relatively potent noncompetitive channel blockers of rat α3β4 nAChRs. Here, we show that these compounds are likewise highly effective blockers of human α3β4 and α4β2 nAChRs. Moreover, we show that they display relatively low affinity for opiate binding sites labeled by [3H]-naloxone. We then evaluated these compounds in rats and mice in preclinical behavioral models predictive of potential anxiolytic and antidepressant efficacy. We found that EMDP, but not EDDP, displayed robust effects predictive of anxiolytic and antidepressant efficacy without significant effects on locomotor activity. Moreover, EMDP at behaviorally active doses, unlike mecamylamine, did not produce eyelid ptosis, suggesting it may produce fewer autonomic side effects than mecamylamine. Thus, the methadone metabolite EMDP may represent a novel therapeutic avenue for the treatment of some affective disorders.

PKCε phosphorylates α4β2 nicotinic ACh receptors and promotes recovery from desensitization.

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. 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. 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. 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.

AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Rat α3β4 Nicotinic Cholinergic Receptors.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3β4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3β4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3β4 and α4β2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3β4 receptors over α4β2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3β4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3β4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat α3β4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4β2 nAChRs. Both α3β4 and α4β2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3β4 receptors than rat α3β4 and human α4β2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3β4 nAChR and shortest for the human α4β2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

Menthol Enhances the Desensitization of Human α3β4 Nicotinic Acetylcholine Receptors.

The α3β4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human α3β4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca(2+) imaging, (86)Rb(+) efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltage-independent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [(3)H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of α3β4 nAChRs by an allosteric action.