Pharmacological and null mutation approaches reveal nicotinic receptor diversity
Introduction
Molecular cloning techniques have identified nine nicotinic acetylcholine receptor subunits (α2–7, β2–4), the mRNAs of which are expressed in varying patterns and quantities throughout the mammalian brain (Lindstrom et al., 1996). Mature nicotinic acetylcholine receptors appear to be pentameric assemblies of these subunits, and because different combinations of subunits produce receptors of different subtypes, the potential for nicotinic acetylcholine receptor diversity in mammalian brain is vast.
Identifying which nicotinic acetylcholine receptor subtypes are expressed in mammalian central nervous system (CNS) has been a difficult task due to a paucity both of truly subtype specific nicotinic compounds, and of well-characterized assays for nicotinic receptor binding and activation. These problems exacerbate each other, and progress in one area is likely to have positive repercussions in the other. To date, extensive biochemical data have only been collected for two native nicotinic acetylcholine receptor subtypes: the “high-affinity agonist binding” α4β2 subtype (labeled by [3H]cytisine and (−)-[3H]nicotine; Whiting and Lindstrom, 1987, Flores et al., 1992, Picciotto et al., 1995, Marubio et al., 1999) and the predominantly or entirely α7 subtype (labeled by [125I]α-bungarotoxin, Schoepfer et al., 1990, Seguela et al., 1992). Identification of additional naturally expressed nicotinic acetylcholine receptor subtypes is a priority as it will yield insights into the rules governing assembly of subunit peptides into receptor proteins, facilitate generation and isolation of subtype specific compounds, and enhance understanding of the physiological roles of individual neuronal nicotinic acetylcholine receptor subtypes in normal and/or pathological states.
One of our laboratory's main priorities has been to develop and characterize binding and functional assays of nicotinic receptors. The primary motivation in this effort has been to identify discrepancies between different measures of nicotinic acetylcholine receptor binding and function, which would indicate heterogeneity in the nicotinic acetylcholine receptor populations responsible.
[3H]Epibatidine has been shown to bind to multiple nicotinic acetylcholine receptor subtypes with high-affinity Perry and Kellar, 1995, Marks et al., 1998, Parker et al., 1998. In rat (Perry and Kellar, 1995) and mouse (Marks et al., 1998) CNS, the majority of high-affinity [3H]epibatidine binding occurs at the α4β2 subtype nicotinic acetylcholine receptor, but additional sites (distinguished by their relatively low cytisine affinity) are also expressed in small nuclei, dispersed across the brain. This information provided the impetus to use [3H]epibatidine binding as a tool to identify novel nicotinic acetylcholine receptor subtypes in mouse brain.
A growing consensus that many nicotinic acetylcholine receptors are located presynaptically, where they modulate neurotransmitter release (Wonnacott, 1997), has encouraged efforts to study nicotinic acetylcholine receptor function in preparations of isolated nerve termini, or synaptosomes. Accordingly, we have concentrated our efforts on developing novel functional assays of nicotinic acetylcholine receptor mediated synaptosomal release. Such assays may be divided into those that monitor neurotransmitter release, an indirect measure of nicotinic acetylcholine receptor activation, and those that measure ion flux through the nicotinic acetylcholine receptor directly. In the first category, we have described nicotinic acetylcholine receptor-mediated mouse brain synaptosomal [3H]dopamine (Grady et al., 1992) and [3H]γ-aminobutyric acid ([3H]GABA) (Lu et al., 1998) efflux assays. In order to directly measure nicotinic acetylcholine receptor-mediated ion flux, we have characterized 86Rb+ efflux assays, using both discrete sampling (Marks et al., 1993) and continuous flow monitoring detection, which offers considerable increases in temporal resolution (Marks et al., 1999). Ion flux assays offer a means to measure activation of all of the nicotinic acetylcholine receptors in a given preparation, while neurotransmitter release assays will only measure activation of nicotinic acetylcholine receptors associated with synaptosomes containing the transmitter in question. Whether this added selectivity is an advantage depends on the particular experimental application. Detailed pharmacological comparisons among these biochemical assays indicate considerable heterogeneity in the nicotinic acetylcholine receptor-mediated responses, suggesting mediation by a number of different nicotinic acetylcholine receptor subtypes.
As an adjunct to the conventional pharmacological approach, we have begun to use subunit null mutant animals, as a further tool to provide insights as to the identities of these putative nicotinic acetylcholine receptor subtypes. Changes in, or losses of, nicotinic measures upon alterations in nicotinic acetylcholine receptor subunit gene expression powerfully implicate that gene's product as a component of the nicotinic acetylcholine receptor mediating the tested measure. The results reported here demonstrate the utility of β2-null mutant mice in establishing the role of the β2 nicotinic acetylcholine receptor subunit in binding and functional measures of mouse brain nicotinic acetylcholine receptors.
Section snippets
Materials
[7,8-3H]Dopamine (40–60 Ci/mmol), (−)-[N-methyl-3H]nicotine (75 Ci/mmol), [125I]α-bungarotoxin (initial specific activity 200 Ci/mmol) and [3H]GABA (84–90 Ci/mmol) were obtained from Amersham, Arlington Heights, IL. [3H]Epibatidine (33.8 Ci/mmol), and carrier free 86RbCl were bought from DuPont-NEN, Boston, MA. The following compounds were purchased from Research Biochemicals International, Natick, MA: (+)-epibatidine hydrochloride, (−)-epibatidine hydrochloride, (+)-anatoxin-a, epiboxidine,
Autoradiography
The nicotinic ligands (−)-[3H]nicotine, [3H]epibatidine, and [125I]α-bungarotoxin exhibited considerable variation in their binding patterns at the levels of the superior and inferior colliculi, as illustrated in Fig. 1. (−)-[3H]Nicotine binding was widespread at the level of the superior colliculus in wild-type animals. Particularly high densities of (−)-[3H]nicotine binding were seen in the whole of the interpeduncular nucleus, and the superior colliculus. The thalamus and some layers of the
Discussion
The assays described in this study represent the results of an effort to develop and characterize a variety of nicotinic acetylcholine receptor-mediated binding and functional measures. The main motivation for this effort was to attempt to detect differences among the assays, pointing to underlying nicotinic acetylcholine receptor diversity. Success in this endeavor would be an important step towards identifying the expression and physiological roles of the potentially wide variety of mammalian
Acknowledgements
This work was supported by grants DA-03194 and DA-11156 from the National Institute on Drug Abuse. ACC is supported, in part, by Research Scientist Award DA-00197 from the National Institute on Drug Abuse.
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