Α4β2 Neuronal Nicotinic Acetylcholine Receptors Research Articles

Discovery of Novel α4β2 Neuronal Nicotinic Receptor Modulators through Structure-Based Virtual Screening.

We performed a hierarchical structure-based virtual screening utilizing a comparative model of the human α4β2 neuronal nicotinic acetylcholine receptor (nAChR) extracellular domain. Compounds were selected for experimental testing based on structural diversity, binding pocket location, and standard error of the free energy scoring function used in the screening. Four of the eleven in silico hit compounds showed promising activity with low micromolar IC50 values in a calcium accumulation assay. Two of the antagonists were also proven to be selective for human α4β2 vs human α3β4 nAChRs. This is the first report of successful discovery of novel nAChR antagonists through the use of structure-based virtual screening with a human nAChR homology model. These compounds may serve as potential novel scaffolds for further development of selective nAChR antagonists.

Potentiation of analgesic efficacy but not side effects: Co-administration of an α4β2 neuronal nicotinic acetylcholine receptor agonist and its positive allosteric modulator in experimental models of pain in rats

Positive modulation of the neuronal nicotinic acetylcholine receptor (nAChR) α4β2 subtype by selective positive allosteric modulator NS-9283 has shown to potentiate the nAChR agonist ABT-594-induced anti-allodynic activity in preclinical neuropathic pain. To determine whether this benefit can be extended beyond neuropathic pain, the present study examined the analgesic activity and adverse effect profile of co-administered NS-9283 and ABT-594 in a variety of preclinical models in rats. The effect of the combined therapy on drug-induced brain activities was also determined using pharmacological magnetic resonance imaging. In carrageenan-induced thermal hyperalgesia, co-administration of NS-9283 (3.5 μmol/kg, i.p.) induced a 6-fold leftward shift of the dose–response of ABT-594 (ED 50 = 26 vs. 160 nmol/kg, i.p.). In the paw skin incision model of post-operative pain, co-administration of NS-9283 similarly induced a 6-fold leftward shift of ABT-594 (ED 50 = 26 vs. 153 nmol/kg). In monoiodo-acetate induced knee joint pain, co-administration of NS-9283 enhanced the potency of ABT-594 by 5-fold (ED 50 = 1.0 vs. 4.6 nmol/kg). In pharmacological MRI, co-administration of NS-9283 was shown to lead to a leftward shift of ABT-594 dose–response for cortical activation. ABT-594 induced CNS-related adverse effects were not exacerbated in presence of an efficacious dose of NS-9283 (3.5 μmol/kg). Acute challenge of NS-9283 produced no cross sensitization in nicotine-conditioned animals. These results demonstrate that selective positive allosteric modulation at the α4β2 nAChR potentiates nAChR agonist-induced analgesic activity across neuropathic and nociceptive preclinical pain models without potentiating ABT-594-mediated adverse effects, suggesting that selective positive modulation of α4β2 nAChR by PAM may represent a novel analgesic approach.

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.

Inhibition of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Volatile Aromatic Anesthetics Depends on Drug Hydrophobicity

Volatile aromatic compounds such as benzene are general anesthetics that cause amnesia, hypnosis, and immobility in response to noxious stimuli when inhaled. Although these compounds are not used clinically, they are frequently found in commercial items such as solvents and household cleaning products and are abused as inhalant drugs. Volatile aromatic anesthetics are useful pharmacological tools for probing the relationship between chemical structure and drug activity at putative general anesthetic targets. Neuronal nicotinic acetylcholine (nACh) receptors are ligand-gated ion channels widely expressed in the brain, which are thought to play important roles in learning and memory. In this study, we tested the hypothesis that aromatic anesthetics reversibly inhibit alpha(4)beta(2) neuronal nACh receptor function and sought to determine the structural correlates of receptor inhibition. Electrophysiological techniques were used to quantify the effects of 8 volatile aromatic anesthetics on currents elicited by 1 mM ACh and mediated by human alpha(4)beta(2) nACh receptors expressed in Xenopus oocytes. All of the volatile aromatic anesthetics used in this study reversibly inhibited alpha(4)beta(2) nACh receptors with IC(50) values ranging from 0.00091 atm for 1,2-difluorobenzene to 0.045 atm for hexafluorobenzene. With the exception of hexafluorobenzene, all of the compounds had IC(50) values less than minimum alveolar concentration. Inhibitory potency correlated poorly with the cation-pi binding energies of the compounds (r(2) = 0.48, P = 0.059). However, there was a good correlation between inhibitory potency and the octanol/gas partition coefficient (r(2) = 0.87, P = 0.0008). Volatile aromatic anesthetics potently and reversibly inhibit human alpha(4)beta(2) neuronal nACh receptors. This inhibition may play a role in producing amnesia. In contrast to N-methyl-d-aspartate receptors, the inhibitory potencies of aromatic anesthetics for alpha(4)beta(2) neuronal nACh receptors seem to be dependent on drug hydrophobicity rather than electrostatic properties. This implies that the volatile aromatic anesthetic binding site in the alpha(4)beta(2) neuronal nACh receptor is hydrophobic in character and differs from the nature of the binding site in N-methyl-D-aspartate receptors.

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [ 125I]epibatidine and 5[ 125I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [ 125I]epibatidine and 5[ 125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [ 125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109–P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [ 125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [ 125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers

( S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human α7 and α4β2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide ( 4) (vs [ 3H]cytisine) was the lowest at α4β2 nAChR ( K i = 10 μM), and predicentrine methiodide ( 2) and xanthoplanine iodide ( 3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors ( K i ≈ 1 μM), while the affinity of the diphenolic boldine methiodide ( 1) was intermediate between these values. At homomeric α7 nAChR, xanthoplanine had the highest affinity ( K i = 10 μM) vs [ 125I]α-bungarotoxin while the other three compounds displaced the radioligand with K i values between 15 and 21 μM. At 100 μM, all four compounds inhibited the responses of these receptors to EC 50 concentrations of ACh. The effects of xanthoplanine iodide ( 3) were studied in more detail. Xanthoplanine fully inhibited the EC 50 ACh responses of both α7 and α4β2 nACh receptors with estimated IC 50 values of 9 ± 3 μM (α7) and 5 ± 0.8 μM (α4β2).

Synthesis of Poison-Frog Alkaloids and Their Pharmacological Effects at Neuronal Nicotinic Acetylcholine Receptors

The flexible and efficient enantioselective synthesis of poison-frog alkaloids has been described using the highly stereoselective conjugate addition reactions as the key step. Several 5,8-disubstituted indolizidines and 1,4-disubstituted quinolizidines have been synthesized according to this strategy. Furthermore, 5,6,8-trisubstituted indolizidne type of poison-frog alkaloid 223A and unique tricyclic poison-frog alkaloid 205B have also been synthesized by sequential use of the above key conjugate addition reaction. Investigations of inhibitory effects of synthetic poison-frog alkaloids on neuronal nicotinic acetylcholine receptors have been conducted, and we found that most of the synthetic compounds showed inhibitory effects on the neuronal nicotinic acetylcholine receptors. Especially, the 5,8-disubstituted indolizidine 235B inhibited the α4 β2-neuronal nicotinic acetylcholine receptors in highly subtype-selective manner. These results suggested that the synthetic alkaloid 235B is a promising lead compound for the drugs designed to treat cholinergic disorders such as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE).

QSAR study of a large set of 3-pyridyl ethers as ligands of the α4β2 nicotinic acetylcholine receptor

Extensive 3D-QSAR studies were performed on 158 diverse analogues of 3-pyridyl ethers, which are excellent ligands of α4β2 neuronal nicotinic acetylcholine receptor (NnAChR). Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques were used to relate the binding affinities with the ligand structures. Two QSAR models were obtained using CoMFA and CoMSIA techniques. The two QSAR models were proved to be statistically significant and have high predictive power. The best CoMFA model yielded the cross-validated q 2 = 0.605 and the non-cross-validated r 2 = 0.862. The derived model indicated the importance of steric (85.9%) as well as electrostatic (14.1%) contributions. The CoMFA model demonstrated the steric field as the major descriptor of the ligand binding. The best CoMSIA model gave q 2 = 0.723 and r 2 = 0.685. This model showed that steric (30.3%) and H-bond interaction (61.8%) properties played major roles in ligand binding process. The squares of correlation coefficient for external test set of 28 molecules were 0.723 and 0.685 for the CoMFA model and the CoMSIA model, respectively. The two models were further graphically interpreted in terms of field contribution maps. SAR studies were also performed on different series of compounds in order to get a more reasonable understanding of the interactions between the ligands and the receptor. With the results, we have also presumed some assistant elements as supplements to the traditional pharmacophoric elements. A crude vision of ligand localization in the ligand-binding pocket of the receptor was also obtained, which would favor for the docking study of this kind of ligands.

Synthesis and radiosynthesis of [ 18F]FPhEP, a novel α 4β 2-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors

FPhEP ( 1, (+/−)-2- exo-(2′-fluoro-3′-phenyl-pyridin-5′-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3′-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain α4β2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP ( 1, K i of 0.24 nM against [ 3H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives ( 3a, b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields = 17% for 1, 9% for 3a and 8% for 3b). FPhEP ( 1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives ( 3a, b—1 mg) and the activated K[ 18F]F–Kryptofix ® 222 complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [ 18F]FPhEP ([ 18F]- 1, 2.22–3.33 GBq, 66–137 GBq/μmol) was obtained after semi-preparative HPLC (Symmetry ® C18, eluent aq 0.05 M NaH 2PO 4/CH 3CN, 80:20 (v:v)) in 75–80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [ 18F]fluoride production batch (10–20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity ( K D 660 pM) of [ 18F]FPhEP ([ 18F]- 1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [ 18F]FPhEP ([ 18F]- 1) and [ 18F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [ 18F]FPhEP ([ 18F]- 1) has potential for in vivo imaging nAChRs in the brain with PET.

Synthesis of a [2- Pyridinyl- 18F]-labelled fluoro derivative of (−)-Cytisine as a candidate radioligand for brain nicotinic α4β2 receptor imaging with PET

In recent years, there has been considerable effort to design and synthesize radiotracers suitable for use in Positron Emission Tomography (PET) imaging of the α4β2 neuronal nicotinic acetylcholine receptor (nAChR) subtype. A new fluoropyridinyl derivative of (−)-cytisine ( 1), namely (−)-9-(2-fluoropyridinyl)cytisine ( 3, K i values of 24 and 3462 nM for the α4β2 and α7 nAChRs subtypes, respectively) has been synthesized in four chemical steps from (−)-cytisine and labelled with fluorine-18 ( T 1/2: 119.8 min) using an efficient two-step radiochemical process [(a) nucleophilic heteroaromatic ortho-radiofluorination using the corresponding N-Boc-protected nitro-derivative, (b) TFA removal of the Boc protective group]. Typically, 20–45 mCi (0.74–1.67 GBq) of (−)-9-(2-[ 18F]fluoropyridinyl)cytisine ([ 18F]- 3, 2–3 Ci/μmol or 74–111 GBq/μmol) were easily obtained in 70–75 min starting from a 100 mCi (3.7 GBq) aliquot of a cyclotron-produced [ 18F]fluoride production batch (20–45% non decay-corrected yield based on the starting [ 18F]fluoride). The in vivo pharmacological profile of (−)-9-(2-[ 18F]fluoropyridinyl)cytisine ([ 18F]- 3) was evaluated in rats with biodistribution studies and brain radioactivity monitoring using intracerebral radiosensitive β-microprobes. The observed in vivo distribution of the radiotracer in brain was rather uniform, and did not match with the known regional densities of nAChRs. It was also significantly different from that of the parent compound (−)-[ 3H]cytisine. Moreover, competition studies with (−)-nicotine (5 mg/kg, 5 min before the radiotracer injection) did not reduce brain uptake of the radiotracer. These experiments clearly indicate that (−)-9-(2-[ 18F]fluoropyridinyl)cytisine ([ 18F]- 3) does not have the required properties for imaging nAChRs using PET.

Nonhalogenated anesthetic alkanes and perhalogenated nonimmobilizing alkanes inhibit alpha(4)beta(2) neuronal nicotinic acetylcholine receptors.

The nonhalogenated anesthetic alkanes, cyclopropane and butane, do not enhance gamma-aminobutyric acid-elicited GABAergic currents, suggesting that these agents produce anesthesia via interactions with other molecular targets. Perhalogenated nonimmobilizing alkanes, such as 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane, also fail to enhance GABAergic currents, but display specific behavioral effects that are distinct from those of structurally similar anesthetics. At concentrations predicted to be anesthetic, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane produce amnesia but fail to produce immobility. Neuronal nicotinic acetylcholine (nACh) receptors are sensitive to many anesthetics and are thought to have an important role in learning and memory. We postulated that neuronal nACh receptors might mediate the common amnestic action of nonhalogenated and perhalogenated alkanes. To test the hypothesis that neuronal nACh receptors have a role in mediating the behavioral effects of general anesthetics and nonimmobilizers, we quantified the inhibitory potencies of nonhalogenated anesthetic alkanes and perhalogenated nonimmobilizing alkanes on currents mediated by alpha(4)beta(2) neuronal nACh receptors. Our studies reveal that anesthetics and nonimmobilizers significantly inhibit alpha(4)beta(2) neuronal nACh receptors at concentrations that suppress learning and with potencies that correlate with their hydrophobicities. These results support the hypothesis that alpha(4)beta(2) neuronal nACh receptors mediate the amnestic actions of alkanes but not their immobilizing actions. The results of this study suggest that the immobilizing actions of general anesthetics do not result from the inhibition of alpha(4beta2) neuronal nicotinic acetylcholine receptors. However, the inhibition of neuronal nicotinic acetylcholine receptors may account for the amnestic activities of general anesthetics and nonimmobilizers.

Nonhalogenated Anesthetic Alkanes and Perhalogenated Nonimmobilizing Alkanes Inhibit α4β2 Neuronal Nicotinic Acetylcholine Receptors

The nonhalogenated anesthetic alkanes, cyclopropane and butane, do not enhance γ-aminobutyric acid-elicited GABAergic currents, suggesting that these agents produce anesthesia via interactions with other molecular targets. Perhalogenated nonimmobilizing alkanes, such as 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane, also fail to enhance GABAergic currents, but display specific behavioral effects that are distinct from those of structurally similar anesthetics. At concentrations predicted to be anesthetic, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane produce amnesia but fail to produce immobility. Neuronal nicotinic acetylcholine (nACh) receptors are sensitive to many anesthetics and are thought to have an important role in learning and memory. We postulated that neuronal nACh receptors might mediate the common amnestic action of nonhalogenated and perhalogenated alkanes. To test the hypothesis that neuronal nACh receptors have a role in mediating the behavioral effects of general anesthetics and nonimmobilizers, we quantified the inhibitory potencies of nonhalogenated anesthetic alkanes and perhalogenated nonimmobilizing alkanes on currents mediated by α4β2 neuronal nACh receptors. Our studies reveal that anesthetics and nonimmobilizers significantly inhibit α4β2 neuronal nACh receptors at concentrations that suppress learning and with potencies that correlate with their hydrophobicities. These results support the hypothesis that α4β2 neuronal nACh receptors mediate the amnestic actions of alkanes but not their immobilizing actions.

Ca 2+ permeability of human heteromeric nAChRs expressed by transfection in human cells

The Ca 2+ permeability of the human heteromeric α3β4, α4β2 and α4β4 neuronal nicotinic acetylcholine receptors (nAChRs) was estimated by measuring the fractional Ca 2+ current ( P f) flowing through the ligand-activated receptor-channels. Simultaneous recordings of transmembrane currents and fluorescence transients, using the whole-cell patch-clamp technique combined with fura-2 fluorescence microscopy, were performed in transiently transfected human cells. The human α4β2 nAChR showed a P f value of 2.6%, while the human α3β4 nAChR showed a similar P f value of 2.7%. Conversely, α4β4 nAChR exhibited a P f value (1.5%) significantly smaller than those of both α4β2 and α3β4 nAChRs. In test experiments performed in HEK 293 cells stably expressing rat GluR1 AMPA receptor subunit, we repeated the determination of P f, whose value (3.2%) has previously been reported by others using the same fluorescent dye; and we found a very similar P f value (3.5%). In further test experiments, we found that P f values of chick α3β4 (4.4%) and α4β4 (2.1%) matched those previously reported by us using confocal fluorescence microscopy. Thus, our findings are consistent with those elsewhere reported even using different experimental procedures, giving a strong support to the following sequence of Ca 2+ permeability: h-α3β4>h-α4β2>h-α4β4.

Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes.

1. Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the alpha7 subunit or alpha4 and beta2 subunits expressed in Xenopus laevis oocytes. 2. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human alpha7 and alpha4beta2 nAChRs, with IC(50) values of 20 and 50 microM respectively. Inhibition of the alpha7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the alpha4beta2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both alpha7 (IC(50) value of 122 nM) and alpha4beta2 (IC(50) value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. 3. Ketamine is a noncompetitive inhibitor at both the alpha7 and alpha4beta2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the alpha7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the alpha4beta2 nAChR. 4. Since alpha7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic.

Inefficient cell-surface expression of hybrid complexes formed by the co-assembly of neuronal nicotinic acetylcholine receptor and serotonin receptor subunits

Previous studies have demonstrated that relatively low levels of α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on the cell surface of transfected mammalian cell lines but that surface expression levels can be dramatically up-regulated by co-expression of these subunits with chimeric subunits containing the N-terminal portion of the neuronal nAChR α4 or β2 subunits together with the C-terminal domain of the 5-HT 3A subunit. Recent work has also suggested that the nAChR α4 subunit can co-assemble in a ‘promiscuous’ manner with the serotonin receptor 5-HT 3A subunit to form functional hybrid receptors. In this study we have examined whether co-assembly of either α4 or β2 with 5-HT 3A itself (rather than with the α4/5-HT 3A or β2/5-HT 3A subunit chimeras) can also facilitate cell surface expression of α4 and β2 subunits in transfected mammalian cells. Evidence has been obtained by immunoprecipitation, cell-surface antibody binding and radioligand binding which indicates that the 5-HT 3A can co-assemble with both the α4 and β2 nAChR subunits. We conclude, however, that co-assembly of 5-HT 3A with either α4 or β2 does not result in efficient cell surface expression of the nAChR subunits and that co-assembled hybrid (nAChR subunit + 5-HT 3R subunit) receptor complexes are largely retained within the cell.

Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation

We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates α4β2 and α7 neuronal nicotinic acetylcholine receptors while α3β2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human α4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and α3 sequences thereafter formed acetylcholine receptors with β2 subunits which were as susceptible to nicotine-induced inactivation as wild-type α4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the α3 subunit and the C-terminal parts of the α4 subunit, formed acetylcholine receptors with β2 subunits which were as resistant to nicotine-induced inactivation as wild-type α3β2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the α subunit.

The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms

The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for α, β, γ, δ subunits (the native foetal muscle subunit combination), or with cRNAs for α, β, ε, δ subunits (the native adult muscle subunit combination), or with cRNAs for α4β2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC 50 11.6, 17.4 and 19.1 μM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range −100 to −40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 μM acetylcholine by 10 nM d-tubocurarine was 29±5% (mean±S.E.M.; n=7), and the inhibition by 10 nM pancuronium was 39±6% (mean±S.E.M.; n=8; P>0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 μM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55±5% ( P<0.01; n=12) and 60±4% ( P<0.001; n=10), respectively. Thus the developmental switch from the γ to the ε subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 μM acetylcholine by 71±6% ( n=10) for foetal and 63±5% ( n=4) for adult nicotinic acetylcholine receptors. In the α4β2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 μM acetylcholine (69±6%, n=6) than 10 nM d-tubocurarine (30±5%; n=6; P<0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the β2 subunit with muscle α, ε and δ subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the αβ1εδ subunit consistent with the hypothesis that the β subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.

Regulation of Human α4β2 Neuronal Nicotinic Acetylcholine Receptors by Cholinergic Channel Ligands and Second Messenger Pathways

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.

An amino acid exchange in the second transmembrane segment of a neuronal nicotinic receptor causes partial epilepsy by altering its desensitization kinetics

The α4 subunit of the neuronal nicotinic acetylcholine receptor is the first gene shown to be involved in a human idiopathic epileptic disease. A missense mutation, leading to the replacement of serine 248 by phenylalanine in the second transmembrane segment, had been detected in patients with autosomal dominant nocturnal frontal lobe epilepsy. The properties of the wild type receptor composed of α4 and β2 subunits and the mutant receptor where α4 subunits carried the mutation at serine 248 were compared by means of cDNA manipulation and expression in Xenopus oocytes. The mutant receptor exhibited faster desensitization upon activation by acetylcholine and recovery from the desensitized state was much slower than in the wild type receptor. We conclude that the reported mutation causes seizures via a diminution of the activity of the α4β2 neuronal nicotinic acetylcholine receptor.

Potentiation and inhibition of subtypes of neuronal nicotinic acetylcholine receptors by Pb 2+

Effects of inorganic lead (Pb 2+) on defined subtypes of neuronal nicotinic acetylcholine receptors have been investigated. Voltage clamp experiments have been performed on Xenopus oocytes expressing α3β2, α3β4 and α4β2 neuronal nicotinic acetylcholine receptor subunit combinations. In oocytes expressing the α3β2 subunit combination Pb 2+ enhances the peak amplitude of nicotinic acetylcholine receptor-mediated inward currents evoked by superfusion with 100 μM acetylcholine. At concentrations of 1–250 μM, Pb 2+ potentiates α3β2 receptor-mediated inward current concentration dependently by a factor of 1.1–11.0. Inward currents evoked by low (3 μM) and high (1 mM) concentrations of acetylcholine are potentiated to a similar extent. Conversely, in oocytes expressing the α3β4 subunit combination Pb 2+ inhibits the nicotinic receptor-mediated inward currents evoked with μM acetylcholine. Inhibitory effects are observed in the concentration range of 1 nM–100 μM Pb 2+, but the degree of inhibition varies between oocytes. A similar inhibition of the α4β2 nicotinic receptor-mediated inward current by Pb 2+ indicates that α as well as β subunits are involved in the potentiating and inhibitory effects. Possible reasons for the variation in the inhibitory effects of Pb 2+ on α3β4 and α4β4 nicotinic receptor-mediated inward currents have been investigated and are discussed. The divalent cations Ca 2+ and Mg 2+ potentiate both α3β2 and α3β4 nicotinic receptor-mediated inward currents. The distinct modulation of receptor function by Pb 2+ and by Ca 2+ and Mg 2+ and the dependence of the modulatory effect of Pb 2+ on subunit composition suggest that Pb 2+ interacts with multiple sites on the α and β subunits of neuronal nicotinic acetylcholine receptors.