Beta2-containing Receptors Research Articles

Similar activity of mecamylamine stereoisomers in vitro and in vivo

A previous characterization of mecamylamine stereoisomers using nicotinic acetylcholine receptors expressed in Xenopus oocytes revealed only small differences between the activity of the R and S forms of mecamylamine. However, that work was limited in the breadth of receptor subtypes tested, especially in regard to the discrimination of high and low sensitivity receptors, which differ in the ratios of alpha and beta subunits. We report new data using subunit concatamers, which produce uniform populations of high-sensitivity or low-sensitivity receptors, as well as alpha2, alpha5, and alpha6-containing receptors, which were not studied previously. Consistent with previous studies, we found that beta4-containing receptors were most sensitive to mecamylamine and that the IC50 values for the inhibition of net charge were lower than for inhibition of peak currents. No large differences were seen between the activities of the mecamylamine isomers. Additionally, a previously reported potentiation of high-sensitivity α4β2 receptors by S-mecamylamine could not be reproduced in the oocyte system, even with mutants that had greatly reduced sensitivity to mecamylamine inhibition or when the selective agonist TC-2559 was used. In vivo studies suggested that the R-isomer might be somewhat more potent than the S isomer at blocking CNS effects of nicotine. Although the potency difference was no more than a factor of two, it is consistent with lower LD50 estimates previously reported for the R isomer. Our results significantly extend knowledge of the nicotinic acetylcholine receptor activity profile of mecamylamine and support the hypothesis that these effects are not strongly stereoisomer selective.

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

Nicotine improves sensory processing in schizophrenic individuals, as measured by changes in auditory event-related potentials (ERPs). Nicotine administration also alters ERPs in mice by increasing the amplitude and gating of the P20 ERP component while decreasing the amplitude of the N40 ERP component. Less is known about the role of specific nicotinic acetylcholine receptor (nAChR) subtypes. In this study, we examined whether nAChRs containing the beta2 subunit contribute to nicotine's effects on auditory ERPs. We tested the effect of nicotine in wild-type mice and mice lacking the beta2 nAChR subunit. Mice underwent stereotaxic implantation of stainless steel electrodes located in the CA3 region of the hippocampus, and 50 paired click stimuli were delivered during each drug condition. There was no significant difference in P20 or N40 amplitude or gating between genotypes during the control condition, suggesting that beta2-containing receptors are not essential for the baseline auditory ERP response. Nicotine increased P20 amplitude and enhanced gating in wild-type and beta2 knockout mice, but only decreased N40 amplitude in wild-type mice. There was no effect of nicotine on N40 gating in either genotype. beta2-containing receptors are necessary for nicotine's effects on the N40 component of the mouse auditory ERP. These results suggest that beta2-containing nAChRs modulate sensory processing and may serve as a therapeutic target in schizophrenic individuals.

Subcellular Trafficking, Pentameric Assembly, and Subunit Stoichiometry of Neuronal Nicotinic Acetylcholine Receptors Containing Fluorescently Labeled α6 and β3 Subunits

Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing alpha4, alpha6, beta2, and beta3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing alpha6 and beta3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged beta3 subunits were fully functional compared with receptors with untagged beta3 subunits. We find that beta3- and alpha6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent alpha4 and beta2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of beta3 and alpha6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single beta3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for alpha4- and alpha6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of alpha6- and beta3-containing receptors using TIRF.

Decreased anxiety-like behavior in beta3 nicotinic receptor subunit knockout mice

Nicotine, via a family of nicotinic acetylcholine receptors, elicits many physiological responses, including alterations in anxiety. Studies suggest that the effects of nicotine on anxiety may support smoking behaviors. We reported previously that mice lacking the beta3 nicotinic receptor subunit demonstrate increased activity in the open field arena. Open field activity has been shown to be a composite of anxiety and locomotor activity, behaviors that are both altered by nicotine. We therefore sought to differentiate the role(s) of beta3-containing receptors in anxiety and locomotor activity. Anxiety behaviors were examined in the elevated plus maze, the black/white box and the mirrored chamber. Beta3 null mutant mice demonstrated decreased anxiety with more time spent on the open arm of the elevated plus maze than their wildtype littermates. No significant differences were observed with the black/white box or the mirrored chamber. Levels of the stress hormone, corticosterone, were significantly higher in the beta3 null mutant mice at baseline and following exposure to stress. Increased locomotor activity in the Y-maze was also observed for the beta3 null mutant mice, but only following exposure to stress. These findings strongly suggest that beta3-containing nicotinic receptors influence anxiety and may be critical for the continuation of smoking behaviors.

Species Selectivity of a Nicotinic Acetylcholine Receptor Agonist Is Conferred by Two Adjacent Extracellular β4 Amino Acids that Are Implicated in the Coupling of Binding to Channel Gating

5-(Trifluoromethyl)-6-(1-methyl-azepan-4-yl)methyl-1H-quinolin-2-one (TMAQ) is a novel nicotinic acetylcholine receptor (nAChR) agonist with strong selectivity for beta4-containing receptors. TMAQ also exhibits remarkable species selectivity, being a potent agonist of nAChRs containing the human beta4 subunit but having no detectable agonist activity on nAChRs containing the rat beta4 subunit. With the aim of identifying subunit domains and individual amino acids, which contribute to the species selectivity of TMAQ, a series of chimeric and mutated beta4 subunits has been constructed. Recombinant receptors containing wild-type, chimeric, or mutated beta4 subunits have been examined by radioligand binding, intracellular calcium assays, and electrophysiological recording. Two adjacent amino acids located within the extracellular loop D domain of the beta4 subunit (amino acids 55 and 56) have been identified as playing a critical role in determining the agonist potency of TMAQ. Mutagenesis of these two residues within the rat beta4 subunit to the corresponding amino acids in the human beta4 subunit (S55N and I56V mutations) confers sensitivity to TMAQ. The converse mutations in the human beta4 subunit (N55S and V56I) largely abolish sensitivity to TMAQ. In contrast, these mutations have little or no effect on sensitivity to the nonselective nicotinic agonist epibatidine. Despite acting as a potent agonist of human beta4-containing nAChRs, TMAQ acts as an antagonist of rat beta4-containing receptors. Our experimental data, together with homology models of the rat and human alpha3beta4 nAChRs, suggest that amino acids 55 and 56 may be involved in the coupling of agonist binding and channel gating.

Mouse β-TC6 Insulinoma Cells: High Expression of Functional α3β4 Nicotinic Receptors Mediating Membrane Potential, Intracellular Calcium, and Insulin Release

Nicotine elicited membrane depolarization, elevation of intracellular calcium, rubidium efflux, and release of insulin from mouse beta-TC6 insulinoma cells. Such responses were blocked by the nicotinic antagonist mecamylamine but not by the muscarinic antagonist atropine. Neither the selective alpha4beta2 antagonist dihydro-beta-erythroidine nor the selective alpha7 antagonist methyllycaconitine significantly blocked the nicotine-elicited depolarization or the calcium response. The elevation of intracellular calcium did not occur in calcium-free media, indicating that the increase in intracellular calcium was due to the influx of calcium. The rank order of potency for nicotinic agonists was as follows: epibatidine > nicotine = 3-(azetidinylmethoxy)pyridine (A-85380), cytisine, dimethylphenylpiperazinium (DMPP). Cytisine and DMPP seemed to be partial agonists. The density of nicotinic receptors measured by [3H]epibatidine binding was 7-fold higher in membranes from beta-TC6 cells than in rat brain membranes. No binding of 125I-A-85380 was detected, indicating the absence of beta2-containing receptors. Reverse transcription-polymerase chain reaction analyses indicated the presence of mRNA for alpha3 and alpha4 subunits and beta2 and beta4 subunits in beta-TC6 cells. The binding and functional data suggest that the major nicotinic receptor is composed of alpha3 and beta4 subunits. The beta-TC6 cells thus provide a model system for pharmacological study of such nicotinic receptors.

Long‐term exposure to the new nicotinic antagonist 1,2‐bisN‐cytisinylethane upregulates nicotinic receptor subtypes of SH‐SY5Y human neuroblastoma cells

Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC50=220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3H-epibatidine (3H-Epi; 3-4-fold) or 125I-alpha-bungarotoxin (125I-alphaBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-beta-erythroidine or hexametonium. Immunoprecipitation and immunopurification studies showed that the increase in 3H-Epi-binding receptors was due to the enhanced expression of alpha3beta2 and alpha3beta2beta4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers alpha-conotoxin MII or methyllycaconitine. In conclusion, chronic treatment with CC4 increased the number of nAChRs containing beta2 and alpha7 subunits on the plasma membrane, where they were functionally active. In the case of beta2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of beta2-containing receptor subtypes..

Activation and desensitization of neuronal nicotinic receptors modulate glutamatergic transmission on neonatal rat hypoglossal motoneurons

In the neonate the muscles of the tongue, which are exclusively innervated by the XII cranial nerves originating from the brainstem nucleus hypoglossus, must contract rhythmically in coincidence with breathing, suckling and swallowing. These motor commands are generated by hypoglossal motoneurons excited by glutamatergic inputs. Because in forebrain areas the efficiency of glutamatergic transmission is modulated by neuronal nicotinic receptors (nAChRs), the role and identity of nAChRs within the nucleus hypoglossus of the neonatal rat were explored using an in vitro brainstem slice preparation. This area expressed immunoreactivity for alpha4, alpha7 and beta2 nAChR subunits. Whole-cell patch-clamp recording from hypoglossal motoneurons showed lack of spontaneous cholinergic events mediated by nAChRs even in the presence of a cholinesterase inhibitor. However, pharmacological antagonism of alpha7- or beta2-containing receptors depressed glutamatergic currents arising either spontaneously or by electrical stimulation of the reticular formation. Hypoglossal motoneurons expressed functional nAChRs with characteristics of alpha4beta2 and alpha7 receptor subunits. Such receptors underwent fast desensitization (time constant of 200 ms) with full recovery within 1 min. Low (0.5 microm) concentration of nicotine first facilitated glutamatergic transmission on motoneurons and later depressed it through receptor desensitization. When 0.1 microm nicotine was used, only depression of synaptic transmission occurred, in keeping with the suggestion that nAChRs can be desensitized without prior activation. These results highlight the role of tonic nAChR activity in shaping excitatory inputs to hypoglossal motoneurons, and suggest that nAChR desensitization by ambient nicotine could contribute to disorders of tongue muscle movements.

Opposing Actions of Etomidate on Cortical Theta Oscillations Are Mediated by Different γ-Aminobutyric Acid Type A Receptor Subtypes

Cortical networks generate diverse patterns of rhythmic activity. Theta oscillations (4-12 Hz) are commonly observed during spatial learning and working memory tasks. The authors ask how etomidate, acting predominantly via gamma-aminobutyric acid type A (GABAA) receptors containing beta2 or beta3 subunits, affects theta activity in vitro. To characterize the effects of etomidate, the authors recorded action potential firing together with local field potentials in slice cultures prepared from the neocortex of the beta3(N265M) knock-in mutant and wild type mice. Actions of etomidate were studied at 0.2 microm, which is approximately 15% of the concentration causing immobility ( approximately 1.5 microm). In preparations derived from wild type and beta3(N265M) mutant mice, episodes of ongoing activity spontaneously occurred at a frequency of approximately 0.1 Hz and persisted for several seconds. Towards the end of these periods, synchronized oscillations in the theta band developed. These oscillations were significantly depressed in slices from beta3(N265M) mutant mice (P < 0.05). In this preparation etomidate acts almost exclusively via beta2 subunit containing GABAA receptors. In contrast, no depression was observed in slices from wild type mice, where etomidate potentiates both beta2- and beta3-containing GABAA receptors. At concentrations assumed to cause sedation and amnesia, etomidate depresses theta oscillations via beta2-containing GABAA receptors but enhances these oscillations by acting on beta3 subunit containing receptors. This indicates that the overall effect of the anesthetic reflects a balance between enhancement and inhibition produced by different GABAA receptor subtypes.

The Effects of β3 Subunit Incorporation on the Pharmacology and Single Channel Properties of Oocyte-expressed Human α3β4 Neuronal Nicotinic Receptors

We compared the main properties of human recombinant alpha3beta4beta3 neuronal nicotinic receptors with those of alpha3beta4 receptors, expressed in Xenopus oocytes. beta3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (alpha3beta4) to 46.7 picosiemens (alpha3beta4beta3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of beta3-containing receptors differed little from those of alpha3beta4. The main pharmacological difference in alpha3beta4beta3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 microM) and dihydro-beta-erythroidine (30 microM) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nM on alpha3beta4 and alpha3beta4beta3, respectively. The change in single channel conductance confirms that beta3 replaces a beta4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the beta3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of beta3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.

Developmental expression of heteromeric nicotinic receptor subtypes in chick retina.

Acting through nicotinic acetylcholine receptors (nAChRs), acetylcholine plays an important role in retinal development and the formation of retinal connections to target tissues, but very little is known about the nAChR subtypes expressed in vertebrate retina during neuronal development. We used immunoprecipitation and [3H]epibatidine binding to study the expression of chick retina alpha-bungarotoxin-insensitive heteromeric nAChRs during development and adulthood, and found that it is strictly developmentally regulated, reaching a peak on postnatal day 1. The increase in [3H]epibatidine receptors is caused mainly by an increase in the receptors containing the alpha2, alpha6, beta3, and beta4 subunits. The contribution of beta subunits to [3H]epibatidine receptors significantly changes during development: the beta2 subunit is contained in the majority (84%) of receptors on embryonic day (E) 7 but in only 32% on postnatal day (P) 1, whereas the beta4-containing receptors increase from 22% to 78% during the same period. Using a sequential immunodepletion procedure, we purified the beta2- and beta4-containing subtypes and found that they coassemble with alpha4 and/or alpha3 on E11, and also with the alpha2, alpha6, and beta3 on P1. After the immunodepletion of alpha6-containing receptors, the beta2- and beta4-containing receptors have a very similar pharmacological profile on P1. Parallel immunoprecipitation experiments in other brain areas showed that the developmentally regulated receptors in optic lobe are those containing the alpha2, alpha5, and beta2 subunits and those containing the alpha4 and beta2 subunits, whereas the receptors in forebrain-cerebellum contain the alpha4 and beta2 subunits with or without the alpha5 subunit. These results indicate that there is an increase in receptor heterogeneity and complexity in chick retina during development that is also maintained in adulthood.

Effects of pyridine ring substitutions on affinity, efficacy, and subtype selectivity of neuronal nicotinic receptor agonist epibatidine.

2'-Pyridine ring substituted analogs of epibatidine were assessed for equilibrium binding affinity, functional potency, and efficacy at rat neuronal nicotinic receptors expressed in Xenopus oocytes. Binding affinities were determined in membrane homogenates from oocytes expressing alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, or alpha4beta4. Efficacy (relative to acetylcholine) and potency were measured electrophysiologically with oocytes expressing alpha3beta4, alpha4beta2, and alpha4beta4. Hydroxy, dimethylamino, and trifluoromethanesulfonate analogs had affinities too low for accurate measurement. The bromo analog had affinities 4- to 55-fold greater at beta2 than at beta4-containing receptors, modestly greater efficacy at alpha4beta4 than at alpha4beta2, and 5- to 10-fold greater potency at a4beta4 than at alpha3beta4 or alpha4beta2. The fluoro analog displayed affinities 52- to 875-fold greater at beta2- than at beta4-containing receptors, efficacy at alpha4beta4 receptors 3-fold greater than at alpha4beta2 and alpha3beta4, and was equipotent at all receptors tested. The norchloro analog showed affinities 114- to 3500-fold greater at beta2- than at beta4-containing receptors, 2-fold greater efficacy at alpha4beta2 and alpha4beta4 than at alpha3beta4, and 4- to 5-fold greater potency at alpha4beta4 and alpha3beta4 than at alpha4beta2. The amino analog displayed affinities 10- to 115-fold greater at beta2- than at beta4-containing receptors, 3-fold greater efficacy at alpha3beta4 than at alpha4beta2, and 2- to 4-fold greater potency at alpha3beta4 and alpha4beta4 than at alpha4beta2. Although these compounds displayed a variety of differences in affinity, efficacy, and potency, with one exception (binding affinity and functional potency at alpha4beta4 receptors) there were no significant correlations among these properties.

Haplotypes of four novel single nucleotide polymorphisms in the nicotinic acetylcholine receptor ?2-subunit (CHRNB2) gene show no association with smoking initiation or nicotine dependence

Several types of evidence, including experiments with mice that lack the nicotinic acetylcholine receptor beta2-subunit gene (CHRNB2), have suggested that a beta2-containing nicotinic receptor is necessary for at least some of the reinforcing properties of nicotine. However, sequence variations in CHRNB2 have not been reported, and its role in influencing human smoking behavior and nicotine dependence is not known. We screened most of the introns and exons and found five novel single nucleotide polymorphisms (SNPs). We tested four of these SNPs in three large, carefully selected samples: nonsmokers (n = 317) and regular smokers low levels of nicotine dependence (ND, n = 238), or smokers with high-ND (n = 317). None of the four polymorphisms we tested, nor their estimated haplotypes, were associated with smoking initiation or progression to nicotine dependence.

New insights into the mechanism of action of hypnotics.

Between 1987 and 1989, the different protein subunits that make up the receptor for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were identified. These make up the alpha, beta, gamma and delta families, for each of which exist several subtypes. This receptor is the molecular target of modern hypnotic drugs (i.e. benzodiazepines, zopiclone, zolpidem and zaleplon). In the 10 years that have followed this milestone, significant progress has been made in exploring the molecular mechanisms of hypnotic drug action. Receptor subtype specificity of hypnotics has been explained in terms of differential affinity for receptors containing different alpha subunits, which are expressed in different brain regions. Zolpidem and zaleplon bind preferentially to alpha1-containing receptors, whereas benzodiazepines and zopiclone are aspecific. Different sets of subunits are encoded in contiguous 'cassettes' on the genome, and the transcription of each set appears to be regulated coherently. The predominant GABA(A) receptor composition found in the brain is alpha1beta2gamma2, which are all encoded on human chromosome 5. Targeted gene disruption has provided clues to the physiological functions served by GABA(A) receptors containing different subunits. Receptors containing gamma2 appear to have a vital role in maintaining appropriate central inhibition, beta3-containing receptors may also be important determinants of excitability in certain brain regions, whereas a clear role for alpha5-, alpha6- and gamma3-containing receptors has not yet been established by these techniques. Site-directed mutagenesis has indicated that benzodiazepines bind to a cleft on the GABA(A) receptor surface at the interface between the alpha and gamma subunits. Other drugs (flumazenil, zopiclone, zolpidem) also bind to the a subunit, but interact with amino acids in different binding domains to the benzodiazepines. The molecular mechanism of hypnotic dependence has been explored, and seems to involve downregulation of transcription of the normally prevalent alpha1, beta2 and gamma2 subunits, and the reciprocal upregulation of the expression of rarer subunits. Chronic treatment with hypnotic drugs that may have less dependence potential, such as zopiclone and zolpidem, appears to produce more limited change in GABA(A) receptor subunit expression. These ideas will be important both for designing new hypnotic drugs with a better safety/efficacy profile, and for evaluating more appropriate ways of using the drugs available today.

The role of beta 2-subunit-containing nicotinic acetylcholine receptors in the brain explored with a mutant mouse.

Neuronal nicotinic receptors comprise a family of pentameric oligomers made up of a combination of 10 different subunits. The beta 2 subunit has the widest pattern of expression in the brain and is thus likely to form a significant fraction of neuronal nicotinic receptors. Using mice lacking the beta 2 subunit, we have shown that nAChRs containing this subunit are responsible for most of the high-affinity binding sites for nicotine, cytisine, and epibatidine in the brain. Functional receptors containing the beta 2-subunit are found in the somatodendritic compartment as well as the axonal compartment of neurons. We have examined the contribution of these receptors to the effects of nicotine on the mesolimbic DA system, which mediates the reinforcing properties of many addictive drugs (including nicotine). Submicromolar doses of nicotine, corresponding to the concentrations of nicotine in vivo in self-administration paradigms, increased the firing rate of dopaminergic neurons in vitro in normal mice but not in mice lacking the beta 2 subunit. Consistently, systemic injection of nicotine induced an increase in extracellular dopamine in normal mice but not in mutant mice, and nicotine self-administration was reduced or suppressed in mutant mice. These data support the view that the beta 2-containing receptors are involved in mediating the reinforcing properties of nicotine.

Neuronal Nicotinic Receptor β2 and β4 Subunits Confer Large Differences in Agonist Binding Affinity

We used equilibrium binding analysis to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus laevis oocytes. The alpha4beta2 receptor bound [3H]cytisine with a Kdapp of 0.74 +/- 0. 14 nM. The rank order of Kiapp values of additional nicotinic ligands, determined in competition assays, was cytisine < nicotine < acetylcholine < carbachol < curare. These pharmacological properties of alpha4beta2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (alpha4beta2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [3H]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. Kdapp values for [3H]epibatidine binding were 10 pM for alpha2beta2, 87 pM for alpha2beta4, 14 pM for alpha3beta2, 300 pM for alpha3beta4, 30 pM for alpha4beta2, and 85 pM for alpha4beta4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1, 1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The beta2-containing receptors had consistently higher affinities for these agonists than did beta4-containing receptors. Particularly striking examples are the affinities displayed by alpha2beta2 and alpha2beta4, which differ in 1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and acetylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively. Although smaller differences in affinity could be ascribed to different alpha subunits, the major factor in determining agonist affinity was the nature of the beta subunit.

4-Oxystilbene compounds are selective ligands for neuronal nicotinic alphaBungarotoxin receptors.

1. Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2. MG 624, F3, F3A and F3B inhibited of 125I-alphaBungarotoxin (alphaBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-alphaBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations. 3. We immobilized the alpha7, beta2 and beta4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-alphaBgtx binding to the alpha7-containing receptors with nM affinity, but inhibited 3H-Epi binding to beta2-containing receptors only at very high concentrations (more than 35 microM); their affinity for the beta4-containing receptors was ten times more than for the beta2-containing subtype. 4. Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick alpha7 receptors with an IC50 of respectively 94 and 119 nM. 5. High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 microM) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 microM). 6. In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic alphaBgtx receptors containing the alpha7 subunit.

The amino terminal half of the nicotinic beta-subunit extracellular domain regulates the kinetics of inhibition by neuronal bungarotoxin.

Subtypes of nicotinic receptors previously reported to be unaffected by neuronal bungarotoxin (NBT), including alpha 3 beta 4-containing and muscle type (alpha 1 beta 1 gamma delta) receptors, are shown to be inhibited by this toxin, but with rapid kinetics of onset and recovery. This inhibition is in contrast to the slow and prolonged inhibition of alpha 3 beta 2-containing receptors, suggesting that the beta subunits determine the kinetics of NBT inhibition of alpha 3 receptors. We have coexpressed chimeric beta subunits with alpha 3, and our results show that the first 121 amino acids of the beta subunit extracellular domain are sufficient to regulate the kinetics of NBT inhibition. This domain is also an important determinant of whether cytisine will act as a full agonist or a partial agonist of nicotinic receptors formed with alpha 3.