Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties
Highlights
► A concatamer of human α3 and β4 nAChR subunits was constructed for expression in Xenopus oocytes. ► Insertion of specific subunits was confirmed with selective antagonists and resistant mutants. ► No effects of the α5 D376N SNP were observed on agonist-evoked responses of α3β4α5 receptors. ► Compared to rat, human α3β4 receptors respond more to varenicline and less to cytisine. ► Our data will potentially impact the side-effect liability of agents used for smoking cessation.
Introduction
Early single-channel studies of the nicotinic acetylcholine receptors of autonomic neurons revealed a rich diversity of channel subtypes (Papke, 1993). Although it is now appreciated that, at least in embryonic ganglia, rapidly desensitizing α7-containing nAChRs are of functional importance, blocking α7 receptors in adult animals generally does not impair ganglionic function, and it is likely that the early single-channel studies only detected an array of more slowly desensitizing heteromeric receptor subtypes. We now know, based on recent studies using knockout animals, that ganglionic receptors are primarily assembled as pentameric complexes containing varying arrangements of α3, β2, β4, and α5 subunits (David et al., 2010).
Although ganglionic blockers were the first drugs used clinically to target neuronal nAChR, most current drug development programs intended to target nAChR in the CNS, either for therapeutics or nicotine dependence, view ganglionic receptors containing α3 in various combinations with β2, β4, and α5 as potential sites for off-target side effects. The human α3-β4-α5 genes are in a cluster at chromosomal location 15q24, and recent genome-wide association studies indicated strong correlations between single nucleotide polymorphisms in the α3-β4-α5 gene cluster and risk for both cancer and nicotine dependence (Chen et al., 2009; Stevens et al., 2008). Nicotine addiction and dependence has been clearly linked to α4* and α6* receptors (Wu and Lukas, 2011), and α5 co-assembly into α4* receptors also promotes high sensitivity to nicotine, suggesting a link between nicotine use and α4β2α5 receptors (Kuryatov et al., 2011). However, recent studies have also demonstrated a link between α3β4α5-containing receptors in the medial habenula and nicotine-related behavior, promoting receptors with combinations of these subunits as an alternative target for the development of smoking cessation drugs (Fowler et al., 2011; Frahm et al., 2011; Gallego et al., 2011; Salas et al., 2009).
It has been shown both in vivo (Grady et al., 2009) and in heterologous expression systems (Boulter et al., 1990; Gerzanich et al., 1998) that α3 will form receptors in various combinations with β2, β4, and α5 subunits. However, α3 and β4 subunits readily form functional receptors without additional subunits, and functional effects of α5 co-expression are much more easily detectable in β2-containing than in β4-containing receptors (Gerzanich et al., 1998). Therefore, since most effectively targeted drug development relies on the use of receptors with known subunit composition, we adopted a strategy previously shown to be useful for controlling the subunit composition of α4* receptors (Zhou et al., 2003), by constructing a concatamer of β4 and α3 (β4−6−α3), suitable for co-expression with monomeric α3, β2, β4, or α5 subunits. The β4−6−α3 construct will provide ligand-binding domains with α3−β4 interfaces, so that co-expressed subunit monomers will, with high likelihood, take the fifth position as a structural subunit in the assembled pentamer.
We provide pharmacological validation of hypothesized subunit compositions and characterize the agonist and partial-agonist profiles of the α3β4 receptor subtypes for ACh, nicotine, and the smoking cessation agents, cytisine and varenicline. Cytisine and varenicline have been proposed to have therapeutic utility through potent partial agonist effects on CNS α4-containing receptors. However, it has been a concern that the reportedly high efficacy of these agents on ganglionic α3-containing receptors might be a source of autonomic side effects. We reevaluate those data and show significant differences from the previously reported data based on the use of rodent receptor subtypes and our current studies based on the use of human receptor clones. Additionally, we used the β4−6−α3 construct to study the D376N variant of α5, specifically associated with smoking and cancer risks.
Section snippets
ACh receptor clones
Human nAChR clones were obtained from Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia PA). Alpha3 and β4 were subcloned into the pSGEM vector, obtained from Dr. Michael Hollmann (Ruhr University, Bochum, Germany), which contains Xenopus β-globin untranslated regions to aid Xenopus oocyte expression. Rat nAChR clones were obtained from Dr. Jim Boulter (University of California, Los Angeles).
Concatamer construction
As the C terminus of β4 is of similar length as that of β2, we followed the scheme of Zhou
Experiments confirming the incorporation of specific structural subunits
When β4−6−α3 was expressed alone it was capable of forming functional receptors with properties similar to those formed when was β4−6−α3 co-expressed with β4, suggesting the assembly of α3(2)β4(3) receptors with tethered supernumerary α3 subunits. That is, the ACh concentration–response curves and the recoveries from TMPH and BTMPS responses were similar (data not shown). In order to obtain better control of structural subunit identity, the RNAs for the monomeric constructs were co-expressed at
Discussion
It has been shown both in vivo and in heterologous expression systems that α3 will form receptors in various combinations with β2, β4, and α5 subunits. Unconstrained expression, when all of these subunits are present, results in a heterogeneous population of receptor subtypes both in neurons and in oocytes. We adopted the strategy of co-expressing a concatamer of β4 and α3 (β4−6−α3), with monomeric β2, β4, or α5 subunits and were able to confirm that we obtained pharmacologically distinct
Conclusion
Receptors containing α3β4 nAChR subunits have long been considered strictly off target for nAChR targeting CNS therapeutics. However, as new roles are being discovered for these receptors in the CNS, in regard to nicotine use and appetite control (Mineur et al., 2011), it becomes important to characterize the α3β4 receptor subtypes and understand how to target them selectively. Therefore programs are being developed for high throughput screening of α3β4-containing receptors, and the use of the
Acknowledgments
We thank Shehd Abdullah Abbas Al Rubaiy, Sara Copeland, Matthew Kimbrell, and Matthew Isaacson for conducting OpusXpress experiments and Dr. Lynn Wecker (University of South Florida) for the use of an additional OpusXpress 6000. Supported by a supplement to GM57481 and James and Esther King Biomedical Research award 1KG12.
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