Pharmacology of alpha-conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice

Subtypes of nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds alpha-conotoxin MII (alpha-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the alpha6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of alpha6beta2* nAChRs expressed in vivo for assessing pharmacology of alpha6beta2* nAChRs. Binding to striatal membranes and function in synaptosomes from (alpha4-/-)(beta3+/+) and (alpha4-/-)(beta3-/-) mice were measured and compared with wild-type (alpha4+/+)(beta3+/+) mice. Gene deletions (alpha4 and beta3) decreased binding of (125)I-alpha-CtxMII without affecting affinity for alpha-CtxMII or inhibition of alpha-CtxMII binding by epibatidine or nicotine. Deletion of the alpha4 subunit substantially increased EC(50) values for both nicotine- and cytisine-stimulated alpha-CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC(50) values was seen upon the additional deletion of the beta3 subunit. The data indicate that one alpha-CtxMII-sensitive nAChR subtype, prevalent on wild-type dopaminergic terminals, has the lowest EC(50) for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of alpha6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.     

Nigrostriatal damage preferentially decreases a subpopulation of alpha6beta2* nAChRs in mouse, monkey, and Parkinson's disease striatum

Parkinson's disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the alpha6beta2(*) nicotinic acetylcholine receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum ((*)denotes the presence of possible additional subunits). In this study, we used a novel alpha-conotoxin MII (alpha-CtxMII) analog E11A to further investigate alpha6beta2(*) nAChR subtypes in mouse, monkey, and human striatum. Receptor competition studies with (125)I-alpha-CtxMII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct alpha6beta2(*) nAChR subtypes. These include a very high (femtomolar) and a high (picomolar) affinity site, with approximately 40% of the sites in the very high affinity form. It is noteworthy that only the high-affinity form was detected in alpha4 nAChR-null mutant mice. Because (125)I-alpha-CtxMII binds primarily to alpha6alpha4beta2beta3 and alpha6beta2beta3 nAChR subtypes in mouse striatum, these data suggest that the population lost in the alpha4 knockout mice was the alpha6alpha4beta2beta3 subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal alpha6beta2(*) populations in two animal models and in Parkinson's disease. There was a preferential loss of the very high affinity subtype in striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), monkeys treated with MPTP, and patients with Parkinson's disease. These data suggest that dopaminergic terminals expressing the alpha6alpha4beta2beta3 population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with alpha-CtxMII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson's disease.     

Expression of nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by beta 3 subunit gene deletion

mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) alpha6 and beta3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that alpha6- or beta3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of beta3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that beta3-null mutation selectively reduced striatal alpha6* nAChR expression by 76% versus beta3(+/+) control. Parallel experiments showed a smaller reduction in both midbrain alpha3* and alpha6* nAChRs (34 and 42% versus beta3(+/+) control, respectively). Sedimentation coefficient determinations indicated that residual alpha6* nAChRs in beta3(-/-) striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified beta3* nAChRs demonstrated that almost all wild-type striatal beta3* nAChRs also contain alpha4, alpha6, and beta2 subunits, although a small population of non-beta3 alpha6* nAChRs is also expressed. beta3 subunit incorporation seemed to increase alpha4 participation in alpha6beta2* complexes. (125)I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of alpha6* nAChRs from the striata of beta3(-/-) mice was similar to those isolated from beta3(+/+) animals. Together, the results of these experiments show that the beta3 subunit is important for the correct assembly, stability and/or transport of alpha6* nAChRs in dopaminergic neurons and influences their subunit composition. However, beta3 subunit expression is not essential for the expression of alpha6*, high-affinity alpha-CtxMII binding nAChRs.     

Long-term nicotine treatment differentially regulates striatal alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR expression and function

Nicotine treatment has long been associated with alterations in alpha4beta2(*) nicotinic acetylcholine receptor (nAChR) expression that modify dopaminergic function. However, the influence of long-term nicotine treatment on the alpha6beta2(*) nAChR, a subtype specifically localized on dopaminergic neurons, is less clear. Here we used voltammetry, as well as receptor binding studies, to identify the effects of nicotine on striatal alpha6beta2(*) nAChR function and expression. Long-term nicotine treatment via drinking water enhanced nonburst and burst endogenous dopamine release from rat striatal slices. In control animals, alpha6beta2(*) nAChR blockade with alpha-conotoxin MII (alpha-CtxMII) decreased release with nonburst stimulation but not with burst firing. These data in control animals suggest that varying stimulus frequencies differentially regulate alpha6beta2(*) nAChR-evoked dopamine release. In contrast, in nicotine-treated rats, alpha6beta2(*) nAChR blockade elicited a similar pattern of dopamine release with nonburst and burst firing. To elucidate the alpha6beta2(*) nAChR subtypes altered with long-term nicotine treatment, we used the novel alpha-CtxMII analog E11A in combination with alpha4 nAChR knockout mice. (125)I-alpha-CtxMII competition studies in striatum of knockout mice showed that nicotine treatment decreased the alpha6alpha4beta2(*) subtype but increased the alpha6(nonalpha4)beta2(*) nAChR population. These data indicate that alpha6beta2(*) nAChR-evoked dopamine release in nicotine-treated rats is mediated by the alpha6(nonalpha4)beta2(*) nAChR subtype and suggest that the alpha6alpha4beta2(*) nAChR and/or alpha4beta2(*) nAChR contribute to the differential effect of higher frequency stimulation on dopamine release under control conditions. Thus, alpha6beta2(*) nAChR subtypes may represent important targets for smoking cessation therapies and neurological disorders involving these receptors such as Parkinson's disease.     

The subunit composition and pharmacology of alpha-Conotoxin MII-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay

The subunit composition and pharmacology of alpha-Conotoxin MII-binding (alpha-CtxMII) nicotinic acetylcholine receptors (nAChR) was studied by an improved [(125)I]-alpha-CtxMII membrane binding method. This binding method facilitates pharmacological studies that have been difficult to accomplish with [(125)I]-alpha-CtxMII autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding. Binding densities and K(d)-values obtained by this [(125)I]-alpha-CtxMII membrane binding were similar to the values obtained by autoradiography or alpha-CtxMII inhibition of [(125)I]-epibatidine binding, verifying that each of these approaches measures the same nAChR population. Binding results with nAChR subunit-null mutant mice confirm and extend observations from earlier studies: [(125)I]-alpha-CtxMII binding measures two sets of alpha6beta2* nAChR (alpha4alpha6beta2beta3 or alpha6beta2beta3). Most nicotinic agonists and antagonists show monophasic inhibition of [(125)I]-alpha-CtxMII binding, indicating that alpha4alpha6beta2beta3 and alpha6beta2beta3 have similar binding properties. Comparison of the binding and activation profiles of alpha6beta2* nAChR to those of other nAChR subtypes (alpha4beta2* and beta4*) indicates that these receptors have distinctly different pharmacology indicating that it may be possible to target alpha6beta2* nAChR selectively to develop compounds that might be therapeutically useful.     

Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice

Pharmacological evaluation of nicotine-stimulated dopamine release from striatum has yielded data consistent with activation of a single population of nicotinic acetylcholine receptors (nAChR). However, discovery that alpha-conotoxin MII (alpha-CtxMII) partially inhibits the response indicates that two classes of presynaptic nAChRs mediate dopamine release. We have investigated the pharmacology and subunit composition of these two classes of nAChR. Inhibition of nicotine-stimulated dopamine release from mouse striatal synaptosomes by alpha-CtxMII occurs within minutes; recovery is slow. The IC50 is 1 to 3 nM. alpha-CtxMII-sensitive and -resistant components have significant differences in pharmacology. The five agonists tested were more potent at activating the alpha-CtxMII-sensitive nAChRs; indeed, this receptor is the highest affinity functional nAChR found, so far, in mouse brain. In addition, cytisine was more efficacious at the alpha-CtxMII-sensitive sites. Methyllycaconitine was 9-fold more potent at inhibiting the alpha-CtxMII-sensitive sites, whereas dihydro-beta-erythroidine was a 7-fold more potent inhibitor of the alpha-CtxMII-resistant response. Both the transient and persistent phases of nicotine-stimulated dopamine release were partially inhibited by alpha-CtxMII with equal potency. The subunit composition of functional nAChRs, was assessed in mice with null mutations for individual nAChR subunits. The beta2 subunit is an absolute requirement for both classes. In contrast, deletion of beta4 or alpha7 subunits had no effect. The alpha-CtxMII-sensitive response requires beta3 and is partially dependent upon alpha4 subunits, probably alpha6beta3beta2 and alpha4alpha6beta3beta2, whereas the alpha-CtxMII-resistant release requires alpha4 and is partially dependent upon alpha5 subunits, probably alpha4beta2 and alpha4alpha5beta2.     

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.     

Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA

Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-alpha-bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.     

Low concentrations of nicotine differentially desensitize nicotinic acetylcholine receptors that include α5 or α6 subunits and that mediate synaptosomal neurotransmitter release

Desensitization is a complex property of nicotinic acetylcholine receptors (nAChR). Several subtypes of nAChR have high sensitivity to nicotine and mediate effects of nicotine at concentrations found in blood of tobacco smokers. Desensitization of some of these receptor subtypes has been studied in model systems, however, other subtypes have been difficult to express heterologously in native forms. In addition, model systems may not have the same accessory molecules and post-translational modifications found in native populations. We have used wild-type and subunit null mutant mice to study desensitization properties of the high sensitivity α4β2-nAChRs including those that have α5 subunits at both GABAergic and dopaminergic nerve terminals. In addition, we have studied the desensitization of one subtype of α6β2-nAChRs at dopaminergic terminals using α4 subunit null mutant mice. Exposure to low nicotine concentrations, leads to rapid, but partial desensitization of activity mediated by these receptors. α4β2-nAChRs including α5 subunits show faster rates of recovery from desensitization than α4β2-nAChRs without α5. Inclusion of the α5 subunit significantly shifts the concentration response for desensitization to higher values, indicating that receptors with α5 subunits are less desensitized by a 10-min exposure to low concentrations of nicotine. Receptors with α6 subunits appear to desensitize to a lesser degree than those with α4 subunits, indicating that α6β2-nAChRs are somewhat resistant to desensitization by nicotine. These results highlight the importance of studying various receptor subtypes in native systems and how they may differentially respond to nicotine and to nicotinic drugs.