Null mutation of the alpha4 nicotinic receptor subunit increases the propensity of muscarinic-mediated neuronal bursting in mouse hippocampal slices

Alpha4 subunit nicotinic cholinergic receptor (nAChR) knock out mice (KO) have a greater susceptibility to proconvulsant-induced seizures than do wild type (WT). The underlying mechanisms remain obscure. We tested whether such seizure-like activity was reflected in bursting activity of hippocampal neurons by recording with intracellular microelectrodes from CA1 pyramidal neurons in slices from WT and KO mice. Intriguingly, while carbachol-induced bursting activity occurred in only 21% of WT slices, qualitatively identical patterns of bursting occurred in 72% of KO slices. Extracellular recordings from CA1 and CA3 regions suggest that carbachol-mediated population activity was regionalized in our preparations. The relative weighting of excitatory to inhibitory synaptic potentials was similar between WT and alpha4 KO mice. However, burst-firing cells had a smaller input time constant than non-bursters. Low-concentration DHbetaE (selective alpha4beta2 nAChR antagonist) did not increase the propensity of WT slices to burst-fire, indicating that absence of alpha4 subunits per se, cannot explain the differences in activity between slices from WT and KO mice. These observations suggest that alpha4 nAChRs are unlikely to be involved in modulating the pattern of bursting neural activity, but their absence could induce subtle developmental changes in the sensitivity of hippocampal circuits to develop this behaviour.     

Mice lacking the alpha4 nicotinic receptor subunit fail to modulate dopaminergic neuronal arbors and possess impaired dopamine transporter function

Neuronal nicotinic acetylcholine receptors (nAChRs) at presynaptic sites can modulate dopaminergic synaptic transmission by regulating dopamine (DA) release and uptake. Dopaminergic transmission in nigrostriatal and mesolimbic pathways is vital for the coordination of movement and is associated with learning and behavioral reinforcement. We reported recently that the D2 DA receptor plays a central role in regulating the arbor size of substantia nigra dopaminergic neurons. Given the known effects of nAChRs on dopaminergic neurotransmission, we assessed the ability of the alpha4 nAChR subunit to regulate arbor size of dopaminergic neurons by comparing responses of wild-type and alpha4 nAChR subunit knockout [alpha4(-/-)] mice to long-term exposure to cocaine, amphetamine, nicotine, and haloperidol, and after substantia nigra neurotoxic lesioning. We found that dopaminergic neurons in adult drug-naive alpha4(-/-) mice had significantly larger terminal arbors, and despite normal short-term behavioral responses to drugs acting on pre- and postsynaptic D2 DA receptors, they were unable to modulate their terminal arbor in response to pharmacological manipulation or after lesioning. In addition, although synaptosome DA uptake studies showed that the interaction of the D2 DA receptor and the dopamine transporter (DAT) was preserved in alpha4(-/-) mice, DAT function was found to be impaired. These findings suggest that the alpha4 subunit of the nAChR is an independent regulator of terminal arbor size of nigrostriatal dopaminergic neurons and that reduced functionality of presynaptic DAT may contribute to this effect by impairing DA uptake.     

Electroencephalographic characterisation of pentylenetetrazole-induced seizures in mice lacking the alpha 4 subunit of the neuronal nicotinic receptor

Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) is associated in some kindreds with mutations in the genes encoding the alpha 4 or beta 2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR). Functional characterisation of the described ADNFLE mutations in oocyte preparations has produced conflicting results, with some studies suggesting hypofunction but others showing increased ligand sensitivity or delayed desensitisation. Knockout mice were studied to investigate extreme hypofunction of alpha 4 nAChRs in vivo. Mutant (Mt) and control mice underwent epidural electroencephalographic (EEG) recording for 2 h in the untreated state and for 1 h following administration of the gamma-amino butyric acid (GABA) antagonist, pentylenetetrazole (PTZ, 80 mg/kg). No spontaneous seizures occurred and no EEG differences were observed between the genotypes in drug na?ve mice. Following PTZ, however, Mt mice showed markedly increased mortality compared to controls (85 vs 30%, P<0.001). Mts also had a greater number of generalised clonic seizures in the first 40 min following injection. In the same period, the EEGs of Mt mice showed an excess of spikes (P=0.033), multi-spike complexes (P=0.002) and continuous fast activity (P=0.017) compared to controls. These findings demonstrate that intact alpha 4 nAChR subunits provide significant in vivo protection against the proconvulsant effects of GABA antagonism.     

Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors

We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors.     

Relating neuronal nicotinic acetylcholine receptor subtypes defined by subunit composition and channel function

Neuronal nicotinic acetylcholine receptors (nAChRs) are widespread, diverse ion channels involved in synaptic signaling, addiction, and disease. Despite their importance, the relationship between native nAChR subunit composition and function remains poorly defined. Chick ciliary ganglion neurons express two major nAChR types: those recognized by alpha-bungarotoxin (alphaBgt), nearly all of which contain only alpha7 subunits (alpha7-nAChRs) and those insensitive to alphaBgt, which contain alpha3, alpha5, beta4, and, in some cases, beta2 subunits (alpha3*-nAChRs). We explored the relationship between nAChR composition and channel function using toxins recognizing alpha7 subunits (alphaBgt), and alpha3/beta4 (alpha-conotoxin-AuIB), or alpha3/beta2 (alpha-conotoxin-MII) subunit interfaces to perturb responses induced by nicotine, alpha7-, or alpha3-selective agonists (GTS-21 or epibatidine, respectively). Using these reagents, fast-decaying whole-cell current components were attributed solely to alpha7-nAChRs, and slow-decaying components mostly to alpha3*-nAChRs. In outside-out patches, nicotine activated brief 60- and 80-pS single nAChR channel events, and mixed-duration 25- and 40-pS nAChR events. Subsequently, 60- and 80-pS nAChR events and most brief 25- and 40-pS events were attributed to alpha7-nAChRs, and long 25- and 40-pS events to alpha3*-nAChRs. alpha3*-nAChRs lacking beta2 subunits seemed responsible for long 25 pS nAChR events, whereas those containing beta2 subunits mediated the long 40 pS nAChR events that dominate single-channel records. These results reveal greater functional heterogeneity for alpha7-nAChRs than previously expected and indicate that beta2 subunits contribute importantly to alpha3*-nAChR function. By linking structural to functional nAChR subtypes, the findings also illustrate a useful pharmacological strategy for selectively targeting nAChRs.     

Proconvulsant-induced seizures in alpha(4) nicotinic acetylcholine receptor subunit knockout mice

The genetic basis of a number of epilepsy syndromes has been identified but the precise mechanism whereby these mutations produce seizures is unknown. Three mutations of the alpha(4) subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified in autosomal dominant nocturnal frontal lobe epilepsy. In vitro studies of two mutations suggest an alteration of receptor function resulting in decreased ion channel current flow. We investigated the response of alpha(4) nAChR subunit knockout mice to the gamma-aminobutyric acid (GABA) receptor antagonists; pentylenetetrazole (PTZ) and bicuculline (BIC), the glutamate receptor agonist kainic acid (KA), the glycine receptor antagonist strychnine and the K(+) channel blocker 4-aminopyridine (4-AP). Mutant (Mt) mice had a greater sensitivity to PTZ and BIC, with an increase in major motor seizures and seizure-related deaths. Furthermore, Mt mice were more sensitive to KA and strychnine, but the effects were much smaller compared to those seen with the GABA receptor antagonists. Paradoxically, Mt mice appeared to be relatively protected from 4-AP-induced major motor seizures and death.The results show that a functional deletion of the alpha(4) nAChR subunit in vivo is associated with a major increase in sensitivity to GABA receptor blockers.     

Extending the analysis of nicotinic receptor antagonists with the study of alpha6 nicotinic receptor subunit chimeras

Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the alpha6 subunit, a component in nAChRs that mediates some of the reinforcing effects of nicotine, is not easily expressed in systems such as the Xenopus oocyte. Certain aspects of alpha6-containing receptor pharmacology have been studied by using chimeric subunits containing the alpha6 ligand-binding domain. However, these chimeras would not be sensitive to an alpha6-selective channel blocker; therefore we developed an alpha6 chimera (alpha4/6) that has the transmembrane and intracellular domains of alpha6 and the extracellular domain of alpha4. We examined the pharmacological properties of alpha4/6-containing receptors and other important nAChR subtypes, including alpha7, alpha4beta2, alpha4beta4, alpha3beta4, alpha3beta2, and alpha3beta2beta3, as well as receptors containing alpha6/3 and alpha6/4 chimeras. Our data show that the absence or presence of the beta4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-beta-erythroidine is most effective on subtypes containing the alpha4 subunit extracellular domain. Receptors containing the alpha6/4 subunit are sensitive to alpha-conotoxin PIA, while receptors containing the reciprocal alpha4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the alpha4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of alpha6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the alpha4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.     

Structural aspects of high affinity ligands for the alpha 4 beta 2 neuronal nicotinic receptor

Neuronal nicotinic acetylcholine receptors (nAChRs) are a heterogeneous family of related ion channels that are widely distributed throughout the central and peripheral nervous systems. They all share a common architecture of five subunit proteins that combine at the cell surface to create a ligand-gated cation permeable pore. Significant effort is currently being expended by medicinal chemistry teams to synthesize ligands that exhibit selectivity for central over peripheral nAChR subtypes. Within the CNS, multiple nAChR subtypes are recognized, and the discovery of ligands exhibiting selectivity among these subtypes offers an opportunity for the development of novel therapeutic agents. The alpha 4 beta 2 subtype is one of the most abundant nAChR subtypes within the CNS, and has been the primary focus of high affinity ligand design. Nicotine (1), and more recently, epibatidine (2) have served as structural templates for the design of the majority of active compounds. Although the diversity of nAChR ligands is growing, the structural requirements necessary for high affinity binding with the alpha 4 beta 2 receptor remain poorly understood. The putative pharmacophoric elements common to all potent alpha 4 beta 2 ligands include (1) a basic or quaternized nitrogen atom, and (2) a less basic nitrogen or a carbonyl oxygen that presumably interact with electron rich and electron deficient sites on the receptor, respectively. The family of currently known high affinity analogs consists of a diverse array of azacycles containing a basic amine. Several additional basic amine fragments have been identified, including the pyrrolizidine nucleus (exemplified by 8) and the 2-azabicyclo[2.2.1]heptane skeleton (exemplified by 9). In addition, we have found that the furo[2,3-b]pyridine heterocycle (compound 10) serves as useful bioisosteric replacement for the pyridyl substituent of nicotine. A preliminary pharmacophore model is proposed in which a reasonable superposition of the putative pharmacophoric elements of the diverse array of high affinity ligands for the alpha 4 beta 2 nAChR reported herein may be accommodated.     

Potentiation of human alpha4beta2 neuronal nicotinic acetylcholine receptor by estradiol.

The modulation of neurotransmitter receptors by various substances can reflect important physiological mechanisms involved in the regulation of neural function. Furthermore, such substances, in particular specific allosteric modulators, can reveal promising therapeutic targets for diseases of the nervous system. From this perspective, we investigated the effects of the steroid hormone estradiol on human neuronal nicotinic acetylcholine receptors expressed either in Xenopus laevis oocytes or human embryonic kidney cells. Acetylcholine-evoked currents were potentiated both by pre- and coapplications of estradiol in alpha4beta2 and alpha4beta4 receptors, but not in alpha3beta2 or alpha3beta4 receptors. The reversible potentiation of alpha4-containing receptors could be induced within seconds in X. laevis oocytes and at micromolar concentrations of estradiol. The potentiation was greatest for responses evoked by low concentrations of acetylcholine, resulting in an apparent increase of receptor affinity. At the single channel level, estradiol potentiation resulted from an increase in opening probability. Finally, the use of functional chimeric or truncated alpha4 subunits demonstrated that a site at the C-terminal tail of the alpha4 subunit is required for estradiol potentiation. These results suggest the presence of a specific site at the human nicotinic acetylcholine receptor alpha4 subunit through which estradiol can cause an allosteric potentiation of acetylcholine-evoked responses.     

Juvenile stress-induced alteration of maturation of the GABAA receptor alpha subunit in the rat

Profound evidence indicates that GABAA receptors are important in the control of physiological response to stress and anxiety. The alpha subunit type composition contributes significantly to the functional characterization of the GABAA receptors. The alpha2, alpha3, alpha5 subunits are predominately expressed in the brain during embryonic and early postnatal periods of normal rats, whilst alpha1 are most prominent during later developmental stages. In the present study, we examined the long-term effects of juvenile stress on GABA alpha subunit expression in adulthood in the amygdala and hippocampus. We applied the elevated platform stress paradigm at juvenility and used the open-field and startle response tests to assess anxiety level in adulthood. Juvenile stress effects without behavioural tests in adulthood were also examined since previous studies indicated that the mere exposure to these tests might be stressful for rats, enhancing the effects of the juvenile exposure to stress. In adulthood, we quantitatively determined the level of expression of alpha1, alpha2 and alpha3 in the hippocampus and amygdala. Our results indicate that subjecting juvenile stressed rats to additional challenges in adulthood results in an immature-like expression profile of these subunits. To test for potential functional implications of these alterations we examined the effects of the anxiolytic (diazepam) and the sedative (brotizolam) benzodiazepines on juvenile stressed and control rats following additional challenges in adulthood. Juvenile stressed rats were more sensitive to diazepam and less sensitive to brotizolam, suggesting that the alterations in GABA alpha subunit expression in these animals have functional consequences.     

MTSEA potentiates 5-HT3 receptors containing the nicotinic alpha4 subunit

In order to study the subunit composition of 5-HT3 receptors (5-HT3R), we report that (2-aminoethyl)methanethiosulfonate (MTSEA) can enhance the function of both nicotinic ACh receptors (nAChRs) comprised of alpha4/beta2 subunits, and heteromeric channels assembled from serotonin 5-HT3R and alpha4 nAChR subunits. MTSEA has no effect on homomeric 5-HT3 receptor channels.     

A novel human nicotinic receptor subunit, alpha10, that confers functionality to the alpha9-subunit.

We present herein the cloning of the human nicotinic acetylcholine receptor alpha9-ortholog and the identification of a new alpha-like subunit (alpha10) that shares 58% identity with alpha9. Whereas alpha10 fails to produce functional receptors alone, it promoted robust acetylcholine-evoked currents when coinjected with alpha9. The presence of alpha10 modifies the physiological and pharmacological properties of the alpha9 receptor indicating that the two subunits coassemble in a single functional receptor. Fusing the N-terminal domain of alpha9 with the rest of the alpha10-cDNA yielded a functional alpha9:alpha10-chimera that displays the acetylcholine binding properties of alpha9 and ionic pore characteristics of alpha10-containing receptors. In addition, alpha9- and alpha10-subunit mRNAs show limited similar tissue distribution patterns and are expressed in cochlea, pituitary gland, and keratinocytes. These data suggest that, in vivo, alpha9-containing receptors coassemble with alpha10-subunit.     

Selective effects of a 4-oxystilbene derivative on wild and mutant neuronal chick alpha7 nicotinic receptor

1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.     

Novel and potent 6-chloro-3-pyridinyl ligands for the alpha4beta2 neuronal nicotinic acetylcholine receptor.

1-[(6-Chloro-3-pyridinyl)methyl]-2-imidazolidine (1), the N-desnitro metabolite of the major insecticide imidacloprid, is known to have similar potency to that of (-)-nicotine as an inhibitor of [3H](-)-nicotine binding at the rat recombinant alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR); IC50 values in the present study are 3.8 nM for (-)-nicotine, 6.0 nM for 1, and 155 nM for imidacloprid. Synthesis of new analogues of 1, modified only in the heterocyclic moiety (five-, six-, or seven-membered rings with NH, S, O, and CH2 substituents), gave compounds varying from 4-fold higher potency (2-iminothiazole analogue 10) to >6000-fold less active than (-)-nicotine. Other potent N-[(6-chloro-3-pyridinyl)methyl] compounds are those in which the heterocyclic imine is replaced with pyrrolidine (19) (IC50 9 nM) or trimethylammonium (22) (IC50 18 nM). A novel conversion of (-)-nicotine to its 6-chloro analogue increased the potency 2-fold. These 6-chloro-3-pyridinyl compounds are of interest as novel nAChR probes and potential metabolites of candidate insecticides.     

The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo

Nicotine, acting at pentameric neuronal nicotinic acetylcholine receptors (nAChRs), is the primary addictive component in tobacco. At low doses, it affects attention, learning, memory, anxiety, cardiovascular responses, thermoregulation, and nociception. At high doses, nicotine produces more drastic behaviors and eventually induces tonic-clonic seizures in rodents. In mammals, several subunits of the nAChRs have been cloned, including eight alpha and three beta subunits. To study the physiological role of the alpha 5 subunit, we have generated alpha 5-deficient mice. These mice have a generally healthy appearance and are normal in a standard battery of behavioral tests. However, the sensitivity of alpha 5 mutant mice to nicotine-induced behaviors and seizures is dramatically reduced compared with their wild-type littermates. These animals have a normal brain anatomy and normal levels of mRNA for other nAChR subunits, namely alpha 4, alpha 6, alpha 7, beta 2, and beta 4. In addition, (125)I-epibatidine and [(125)I]alpha-bungarotoxin binding in the brains of alpha 5-deficient mice is normal. Together, these results suggest a direct involvement of the alpha 5 subunit in the observed phenotypes.     

A new look at the neuronal nicotinic acetylcholine receptor pharmacophore

Interest in the field of nicotinic receptors has been recently stimulated both by the discovery of the potential therapeutic effects of new agonists, and by the discovery of an association between nicotinic receptor mutations and human neurological diseases. Expression of human receptors in an exogenous system allows their study in isolation. Receptors reconstituted by pairwise injection of either alpha4 or alpha3 with beta2 or beta4 subunits displayed important differences between the resulting receptor subtypes. These results were further compared with those obtained with alpha3:alpha4 fusion proteins. The modifications of either the ligand-binding site in the N-terminal domain or in the ionic pore domain were found to affect the pharmacological properties of the receptors. Finally, the analysis of non-natural derivatives of epibatidine demonstrates how an agonist can be modified to be selective at one receptor subtype or to become an antagonist. These data are well explained on the basis of a three-state allosteric model.     

Varenicline: an alpha4beta2 nicotinic receptor partial agonist for smoking cessation

Herein we describe a novel series of compounds from which varenicline (1, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine) has been identified for smoking cessation. Neuronal nicotinic acetylcholine receptors (nAChRs) mediate the dependence-producing effects of nicotine. We have pursued alpha4beta2 nicotinic receptor partial agonists to inhibit dopaminergic activation produced by smoking while simultaneously providing relief from the craving and withdrawal syndrome that accompanies cessation attempts. Varenicline displays high alpha4beta2 nAChR affinity and the desired in vivo dopaminergic profile.