Gates and filters: unveiling the physiological roles of nicotinic acetylcholine receptors in dopaminergic transmission.

Neuronal nicotinic acetylcholine receptors (nAChRs) in the brain have been enigmatic players on the cerebral stage. As ligand-gated ion channels they were expected to mediate fast cholinergic transmission, yet their influence appears to be modulatory. Two reviews in this issue of the BJP consider the relationship between nAChRs and endogenous ACh, with respect to the modulation of dopaminergic signalling. In his review, Maskos posits that in midbrain dopamine neurons, somatodendritic nAChRs activated by cholinergic inputs from the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) serve as a 'gate' that facilitates the switch to burst firing. In the terminal field, Exley and Cragg argue that nAChRs function as a 'presynaptic filter' to enhance the contrast between single and repetitive spike firing. Thus somatodendritic and presynaptic nAChRs exert subtle and complementary influences in responding to cholinergic inputs.

Presynaptic nicotinic ACh receptors.

Nicotinic ACh (nACh) receptors in the CNS are composed of a diverse array of subunits and have a range of pharmacological properties. However, despite the fact that they are ligand-gated cation channels, their physiological functions have not been determined. This has led to increased interest in presynaptic nACh receptors that act to modulate the release of transmitter from presynaptic terminals.

UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

The neurotoxin histrionicotoxin interacts with the putative ion channel of the nicotinic acetylcholine receptors in the central nervous system.

Perhydrohistrionicotoxin at micromolar concentrations blocked the nicotine-evoked transmitter release from perfused striatal (dopaminergic) and hippocampal (cholinergic) nerve terminals. Perhydrohistrionicotoxin failed to compete with [3H]nicotine for its high-affinity binding site in rat brain, suggesting that the action of this toxin on central nicotinic receptors is noncompetitive. From the dose-response curve, 50% inhibition of nicotine-evoked striatal dopamine release occurred at 5 microM perhydrohistrionicotoxin, a value similar to that obtained in frog sartorius muscle and Electrophorus electroplax. This close agreement may suggest that the ionic channel of the presynaptic nicotinic acetylcholine receptor of brain neurons has similar properties to those of the peripheral receptor.

Agonist pharmacology of the neuronal alpha 7 nicotinic receptor expressed in Xenopus oocytes.

The potencies and efficacies of seven agonists at chick alpha 7 nicotinic receptors expressed in Xenopus oocytes were determined by whole cell recording. (+)-Anatoxin-a was the most potent agonist (EC50 = 0.58 microM) and acetylcholine was the least potent (EC50 = 320 microM). The rank order of agonist potencies was: (+)-anatoxin-a >> cytisine > (-)-nicotine > (+)-nicotine > DMPP > 1-acetyl-4-methylpiperazine methiodide > acetylcholine. DMPP evoked only very small currents: comparison of maximally effective agonist concentrations showed that DMPP was only one-fifth as efficacious as other agonists. Previously published IC50 values for rat brain [125I]alpha-bungarotoxin sites show a similar agonist profile, and the identity of homo-oligomeric alpha 7 receptors with native alpha-bungarotoxin-sensitive neuronal nicotinic receptors is discussed.

Conversion of the sodium channel activator aconitine into a potent alpha 7-selective nicotinic ligand.

Methyllycaconitine (MLA) is a competitive antagonist of nicotinic acetylcholine receptors, with a remarkable preference for neuronal [125I]alpha Bgt binding sites. We have begun to investigate the structural basis of its potency and subtype selectivity. MLA is a substituted norditerpenoid alkaloid linked to a 2-(methylsuccinimido)benzoyl moiety. Hydrolysis of the ester bond in MLA to produce lycoctonine diminished affinity for rat brain [125I]alpha Bgt binding sites 2500-fold and abolished affinity for [3H]nicotine and muscle [125I]alpha Bgt binding sites. The voltage-gated Na+ channel activator aconitine, also a norditerpenoid alkaloid, but with significant structural differences from lycoctonine, displayed comparable weak or absent nicotinic activity. Addition of a 2-(methylsuccinimido)benzoyl sidechain to O-demethylated aconitine, to mimic MLA, abolished Na+ channel activation and conferred nanomolar affinity for brain [125I]alpha Bgt binding sites, comparable to that of MLA. We propose that the ester-linked 2-(methylsuccinimido)benzoyl group is necessary for nicotinic potency, but alpha 7 selectivity resides in the norditerpenoid core of the molecule.

Methyllycaconitine: a selective probe for neuronal alpha-bungarotoxin binding sites.

The ability of methyllycaconitine (MLA) to inhibit the binding of [125I]alpha-bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (Ki 1.4 x 10(-9) M) than for the muscle receptors (Ki 10(-5)-10(-6) M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their alpha-bungarotoxin-binding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems.

Specific high-affinity binding sites for 125I-alpha-bungarotoxin and (-)-[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I-alpha-bungarotoxin had similar Kd values of 1.5 x 10(-9) and 0.8 x 10(-9) M for rat and locust preparations, respectively; the corresponding values for the (-)-[3H]nicotine-binding site were 9.3 x 10(-9) and 1.7 x 10(-7) M. Methyllycaconitine (MLA) potently inhibited 125I-alpha-bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (-)-[3H]nicotine binding whereas (+)-anatoxin-a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)-anatoxin-a is a useful probe for the high-affinity nicotine-binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds alpha-bungarotoxin.

Nicotinic acetylcholine receptors in cultured neurons from the hippocampus and brain stem of the rat characterized by single channel recording.

Single channel recording techniques have been applied to neurons cultured from the hippocampus and the respiratory area of the brain stem of fetal rats in order to search for nicotinic acetylcholine receptors (nAChR) in the central nervous system. In addition to acetylcholine (ACh), the potent and specific agonist (+)-anatoxin-a was also used to characterize nicotinic channels. nAChRs were concentrated on the somal surface near the base of the apical dendrite, and in some patches their density was sufficient to record 2 or more channel openings simultaneously. Although a multiplicity of conductance states was also evident, the predominant population showed a single channel conductance of 20 pS at 10 degrees C. Thus, these neuronal nAChRs resembled the embryonic or denervated-type nAChRs in muscle. However, channel opening and closing kinetics were faster than reported for similar conductance channels in muscle. Therefore the nicotinic channels described here are similar but not identical to those of the well-characterized muscle nAChR, in agreement with biochemical, pharmacological, and molecular genetic studies on brain AChR.

Presynaptic localisation of the nicotinic acetylcholine receptor beta2 subunit immunoreactivity in rat nigrostriatal dopaminergic neurones.

Nicotinic acetylcholine receptors (nAChR) are widely distributed in the central nervous system, where they exert a modulatory influence on synaptic transmission. For the striatum, pharmacological evidence supports the presence of presynaptic alpha3beta2* and alpha4beta2* nAChR that modulate dopamine release from nigrostriatal terminals. The objective of this study was to examine the precise subcellular distribution of the nAChR beta2 subunit in these neurones and its localisation at presynaptic sites. Double immunolabelling with tyrosine hydroxylase (TH) at the confocal level revealed that the cell bodies and axon terminals (synaptosomes) of nigrostriatal neurones were also immunoreactive for the nAChR beta2 subunit. Double-preembedding electron microscopy confirmed that beta2-immunogold labelling was enriched in TH-positive terminals in the dorsal striatum. Quantitative analysis of doubly immunogold-labelled sections in postembedding electron microscopy showed that 86% of TH-positive axonal boutons are also labelled for the nAChR beta2 subunit, whereas 45% of beta2 subunit-immunolabeled boutons do not contain TH. Thus the beta2 subunit is localised within at least two populations of axon terminals in the dorsal striatum. In these structures, 15% of beta2 subunit immunoreactivity was at the plasma membrane but was rarely associated with synapses. These findings are compatible with functional presynaptic beta2-containing nAChR that may be stimulated physiologically by acetylcholine that diffuses from synaptic or nonsynaptic sites of acetylcholine release. These results demonstrate the presynaptic localisation of an nAChR subunit in nigrostriatal dopaminergic neurones, providing morphological evidence for the presynaptic nicotinic modulation of dopamine release.

Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. Using rat striatal synaptosomes loaded with [3H]dopamine, we have characterized the action of the selective nicotinic agonist, (+/-)anatoxin-a, with respect to [3H]dopamine release, in order to explore the mechanisms coupling nicotinic receptor activation to exocytosis. Anatoxin-a evoked [3H]dopamine release in a concentration-dependent and mecamylamine-sensitive manner, EC50 = 0.11 microM. The maximum [3H]dopamine release elicited by anatoxin-a was only 20% of the maximum elicited by KCl depolarization; there was no additivity between anatoxin-a and sub-maximal concentrations of KCl. Both agents stimulated Ca(2+)-dependent release that was equally sensitive to inhibition by 200 microM Cd2+. This result suggests that anatoxin-a-stimulated exocytosis is mediated by Ca2+ influx via voltage-sensitive Ca2+ channels, with little contribution from Ca2+ entering directly through the nicotinic receptor channel. This view is supported by the abolition of anatoxin-a-evoked [3H]dopamine release in Na(+)-depleted medium. A partial (40%) inhibition by tetrodotoxin was observed. These data suggest that activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage-sensitive Ca2+ and Na+ channels. The latter may then amplify the response, activating further Ca2+ channels. The particular voltage-sensitive Ca2+ channels involved remain to be determined.

Tetrodotoxin-sensitivity of nicotine-evoked dopamine release from rat striatum.

Recent observations from synaptosome preparations have questioned the tetrodotoxin (TTX) insensitivity of nicotine-evoked release in the striatum, a characteristic previously considered diagnostic of presynaptically located nicotinic acetylcholine receptors (nAChRs). Therefore, we have undertaken a comparison of nicotine-evoked dopamine release in the presence of TTX from the rat striatum in vitro, using synaptosomes and brain slices, and in vivo, using microdialysis. In P2 and Percoll-purified synaptosome preparations, 1.5 microM TTX partially inhibited nicotine-evoked [3H]dopamine release by 54% and 37%, respectively, whereas in more intact preparations (brain slices and microdialysis) TTX completely inhibited mecamylamine-sensitive nicotine-stimulated dopamine release. These results suggest that caution should be exercised in the interpretation of TTX sensitivity of nicotine-evoked responses with regard to the location of nAChRs.

The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca(2+) dependent mechanism.

Neuronal nicotinic acetylcholine receptors (nAChR) have been suggested to play a role in a variety of modulatory and regulatory processes, including neuroprotection. Here we have characterized the neuroprotective effects of nicotine against an excitotoxic insult in primary hippocampal cultures. Exposure of hippocampal neurons to 200 microM NMDA for 1 h decreased cell viability by 25+/-5%, an effect blocked by NMDA receptor antagonists. Nicotine (10 microM) counteracted the NMDA-induced cell death when co-incubated with NMDA or when present subsequent to the NMDA treatment. Nicotine protection was prevented by 1 microM MLA, confirming that it was mediated by nAChR, and by 1 microM alpha-bungarotoxin, demonstrating that the alpha7 nAChR subtype was responsible. Both the NMDA evoked neurotoxicity and nicotine neuroprotection were Ca(2+)-dependent. In Fura-2-loaded hippocampal neurons, nicotine (10 microM) and NMDA (200 microM) acutely increased intracellular resting Ca(2+) from 70 nM to 200 and 500 nM, respectively. Responses to NMDA were unaffected by the presence of nicotine. (45)Ca(2+) uptake after a 1 h exposure to nicotine or NMDA also demonstrated quantitative differences between the two drugs. This study demonstrates that the alpha7 subtype of nAChR can support neuronal survival after an excitotoxic stimulus, through a Ca(2+) dependent mechanism that operates downstream of NMDA receptor activation.

Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors.

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for alpha bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [3H]MLA bound to rat brain membranes with high affinity (Kd = 1.86 +/- 0.31 nM) with a good ratio of specific to non-specific binding. The binding of [3H]MLA was characterised by rapid association (t 1/2 = 2.3 min) and dissociation (t 1/2 = 12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with alpha bungarotoxin-sensitive nAChR. The snake alpha-toxins, alpha bungarotoxin and alpha cobratoxin, displaced [3H]MLA with high affinity (Ki = 1.8 +/- 0.5 and 5.5 +/- 0.9 nM, respectively), whereas nicotine was less potent (Ki = 6.1 +/- 1.1 microM). The distribution of [3H]MLA binding sites in crudely dissected rat brain regions was identical to that of [125I] alpha bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [3H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake alpha toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [3H]MLA, therefore, is a novel radiolabel for characterising alpha 7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled alpha bungarotoxin for rapid and accurate equilibrium binding assays.

Nudicauline and elatine as potent norditerpenoid ligands at rat neuronal alpha-bungarotoxin binding sites: importance of the 2-(methylsuccinimido)benzoyl moiety for neuronal nicotinic acetylcholine receptor binding.

Methyllycaconitine (MLA, 1) is a novel, potent probe for mammalian and insect nicotinic acetylcholine receptors (nAChR) and displays remarkable selectivity toward neuronal [125I]-alpha-bungarotoxin (alpha BgTX) binding sites that correspond to alpha 7-type nAChR in mammalian brain. We have shown that, among a number of selected norditerpenoid alkaloids, elatine (2) and nudicauline (3) are equipotent with, or better than, MLA (1) in binding to brain [125I]-alpha BgTX binding sites, with IC50 values of 6.1, 1.7, and 7.6 nM, respectively. The 2-((S)-methylsuccinimido)benzoyl moiety of these ligands is crucial for high-affinity binding, whereas structural modifications to the norditerpenoid core of the ligand can be tolerated without loss of activity or selectivity. In addition to MLA (1), elatine (2), and nudicauline (3), we have examined lycoctonine (4), inuline (6), lappaconitine (7), N-desacetyllappaconitine (8), delsoline (10), delcorine (11), deltaline (12), condelphine (13), and karacoline (14). This study therefore extends the range of norditerpenoids, other than MLA, which can be used to probe this important class of nAChR. All 12 alkaloids were assessed for activity at [3H]nicotine binding sites which are considered to represent alpha 4 beta 2 nAChR. Furthermore, the 1H and 13C NMR spectroscopic data of MLA and elatine have been critically compared.

Immunological cross-reactivity between the alpha-bungarotoxin-binding component from rat brain and nicotinic acetylcholine receptor.

An alpha-bungarotoxin-binding protein was partially purified from rat brain and, when complexed with [125I] alpha-bungarotoxin, was shown to behave as a single radiolabelled protein that is distinct from the similarly complexed nAChR from Torpedo marmorata. The alpha-bungarotoxin-binding protein was used as antigen in radioimmunoassays for rabbit anti-(rat muscle nAChR) and rabbit anti-(Torpedo nAChR) antibodies, giving titres approximately 5% and 0.5%, respectively, of those obtained by using homologous antigen in the same assay.

Involvement of protein kinase C in the presynaptic nicotinic modulation of [(3)H]-dopamine release from rat striatal synaptosomes..

1. Presynaptic nicotinic ACh receptors modulate transmitter release in the brain. Here we report their interactions with protein kinase C (PKC) with respect to [(3)H]-dopamine release from rat striatal synaptosomes, monitored by superfusion. 2. Two specific PKC inhibitors, Ro 31-8220 (1 microM) and D-erythro-sphingosine (10 microM) significantly reduced (by 51 and 26% respectively) [(3)H]-dopamine release stimulated by anatoxin-a (AnTx), a potent and selective agonist of nicotinic ACh receptors. The inactive structural analogue of Ro 31-8220, bisindolylmaleimide V (1 microM) had no effect. 3. Two phorbol esters, PDBu (1 microM) and PMA (1 microM) potentiated AnTx-evoked [(3)H]-dopamine release by 50 - 80%. This was Ca(2+)-dependent and prevented by PKC inhibitors. In the absence of nicotinic agonist, phorbol esters enhanced basal release through a PKC-independent mechanism. 4. A (86)Rb(+) efflux assay of nicotinic ACh receptor function confirmed that Ro 31-8220 has no nonspecific effect on presynaptic nicotinic ACh receptors. 5. These results suggest that PKC is activated by nicotinic ACh receptor stimulation and mediates a component of AnTx-evoked [(3)H]-dopamine release. In addition, independent activation of PKC can further amplify the response, offering a potential mechanism for receptor crosstalk.

Differential effects of chronic drug treatment on alpha3* and alpha7 nicotinic receptor binding sites, in hippocampal neurones and SH-SY5Y cells.

1. The aim of this study was to compare the effects of chronic treatment (for 4 or 7 days) with nicotinic drugs and 20 mM KCl on numbers of surface alpha7 nicotinic AChR, identified by [(125)I]-alpha bungarotoxin (alpha-Bgt) binding, in primary hippocampal cultures and SH-SY5Y cells. Numbers of alpha3* nicotinic AChR were also examined in SH-SY5Y cells, using [(3)H]-epibatidine, which is predicted to label the total cellular population of predominantly alpha3beta2* nicotinic AChR under the conditions used. 2. All the nicotinic agonists examined, the antagonists d-tubocurarine and methyllycaconitine, and KCl, upregulated [(125)I]-alpha Bgt binding sites by 20 - 60% in hippocampal neurones and, where examined, SH-SY5Y cells. 3. Upregulation of [(125)I]-alpha-Bgt binding sites by KCl was prevented by co-incubation with the L-type Ca2+ channel blocker verapamil or the Ca2+-calmodulin dependent kinase II (CaM-kinase II) inhibitor KN-62. Upregulation of [(125)I]-alpha-Bgt binding sites by nicotine or 3,[(4-dimethylamino) cinnamylidene] anabaseine maleate (DMAC) was insensitive to these agents. 4. [(3)H]-Epibatidine binding sites in SH-SY5Y cells were not affected by KCl but were upregulated in a verapamil-insensitive manner by nicotine and DMAC. KN-62 itself provoked a 2 fold increase in [(3)H]-epibatidine binding. The inactive analogue KN-04 had no effect, suggesting that CaM-kinase II plays a role in regulating numbers of alpha3* nicotinic AChR. 5. These data indicate that numbers of alpha3* and alpha7 nicotinic AChR are modulated differently. Nicotinic agonists and KCl upregulate alpha7 nicotinic AChR through distinct cellular mechanisms, the latter involving L-type Ca2+ channels and CaM-kinase II. In contrast, alpha3* nicotinic AChR are not upregulated by KCl. This difference may reflect the distinct physiological roles proposed for alpha7 nicotinic AChR.

Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats.

1. Isoarecolone was approximately 250 times less potent than nicotine as an inhibitor of [3H]-nicotine binding to rat brain membranes. Isoarecolone failed to inhibit the binding of the nicotinic ligand [125I]-alpha-bungarotoxin or of the muscarinic ligand [3H]-QNB. 2. Nicotine (0.01-30 microM) evoked the release of [3H]-dopamine from striatal and frontal cortex synaptosomes, with EC50 values of approximately 0.5 microM in each case. This release was largely mecamylamine-sensitive. 3. Isoarecolone (1-200 microM) evoked predominantly mecamylamine-sensitive dopamine release from both striatal and cortical synaptosomes, with a potency at least 20 times less than that of nicotine. The maximum effect of isoarecolone was less than that of nicotine, particularly in the frontal cortex preparation. 4. In control rats treated chronically with saline, neither nicotine nor isoarecolone had clear effects on locomotor activity at the doses tested. Chronic treatment with nicotine clearly sensitized rats to the locomotor activating effect of isoarecolone was seen at a dose about 40 times larger than that of nicotine. 5. The low potency and efficacy of isoarecolone in facilitating sensitized locomotor activity resembled its lower potency and efficacy, compared with nicotine, in evoking dopamine release in vitro. The agonist profile of the nicotinic receptor population mediating dopamine release may determine the pharmacological characteristics of consequent locomotor behaviour.

Interaction of p-aminophenyldichloroarsine, an arsenical with specificity for vicinal cysteines, with [3H]cytisine binding sites in rat brain membranes.

The arsenical compound p-aminophenyldichloroarsine (APA) is selective for spatially close thiols with which it forms a stable complex. The alpha subunits of nicotinic acetylcholine receptors are defined by the presence of a pair of adjacent cysteines close to the agonist binding site. Here the interaction of APA with [3H]cytisine binding sites, which correspond to the major subtype of nicotinic receptors in rat brain has been examined. Incubation of brain membranes with 10 microM APA abolished [3H]cytisine binding. The action of APA was dependent on prior reduction of sulphydryls with dithiothreitol. APA effects could not be reversed by oxidizing agents but could be reversed by the antiarsenical reagent 2,3-dimercapto-1-propane sulphonic acid. Under the conditions used, the concentration of APA producing a half-maximal decrease in binding was 130 nM. The loss of [3H]cytisine binding was due to a decrease in the number of binding sites (Bmax) with no effect on affinity for the radioligand (Kd). Nicotinic ligands failed to protect against the reduction and arsenylation of neuronal receptor sites. These observations are consistent with the potent interaction of APA with this neuronal nicotinic receptor.