Extending the analysis of nicotinic receptor antagonists with the study of α6 nicotinic receptor subunit chimeras

Abstract

Heterologous expression systems have increased the feasibility of developing selective ligands to target nicotinic acetylcholine receptor (nAChR) subtypes. However, the α6 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 α6-containing receptor pharmacology have been studied by using chimeric subunits containing the α6 ligand-binding domain. However, these chimeras would not be sensitive to an α6-selective channel blocker; therefore we developed an α6 chimera (α4/6) that has the transmembrane and intracellular domains of α6 and the extracellular domain of α4. We examined the pharmacological properties of α4/6-containing receptors and other important nAChR subtypes, including α7, α4β2, α4β4, α3β4, α3β2, and α3β2β3, as well as receptors containing α6/3 and α6/4 chimeras. Our data show that the absence or presence of the β4 subunit is an important factor for sensitivity to the ganglionic blocker mecamylamine, and that dihydro-β-erythroidine is most effective on subtypes containing the α4 subunit extracellular domain. Receptors containing the α6/4 subunit are sensitive to α-conotoxin PIA, while receptors containing the reciprocal α4/6 chimera are insensitive. In experiments with novel antagonists of nicotine-evoked dopamine release, the α4/6 chimera indicated that structural rigidity was a key element of compounds that could result in selectivity for noncompetitive inhibition of α6-containing receptors. Our data extend the information available on prototypical nAChR antagonists, and establish the α4/6 chimera as a useful new tool for screening drugs as selective nAChR antagonists.

Introduction

There are three major classes of nicotinic acetylcholine receptors (nAChR) in mammals: muscle-type receptors, heteromeric neuronal-type receptors, and homomeric neuronal receptors. All nAChRs are believed to form pentameric complexes. Important elements of the agonist binding sites are part of the alpha subunits, but the binding sites are located at the interface between alpha and non-alpha subunits, and the non-alpha subunits contribute importantly to the pharmacological properties of the receptors (Luetje and Patrick, 1991), including β1 in regard to the relative insensitivity of muscle-type receptors to ganglionic blockers (Webster et al., 1999).

Eight alpha subunit genes have been cloned from mammalian neuronal tissue (α2, α3, α4, α5, α6, α7, α9, and α10), as well as three non-alpha subunits (β2, β3, and β4). The simplest of the neuronal nAChR to study in the oocyte expression system is the homomeric subtype, which in mammalian brain is composed of only α7 subunits (Gotti et al., 2006). The heteromeric neuronal-type receptors form as various combinations of alpha and beta subunits, always containing at least one type of alpha (either α2, α3, α4, or α6) and at least one type of beta subunit. In the continued presence of agonist, heteromeric receptors convert to a desensitized state that binds nicotine and other agonists with high affinity; these receptors were first identified in radioligand binding studies with rat brain slices (Clarke et al., 1985).

Although there are many possible combinations of neuronal alpha and beta subunits, it has been shown that the majority of the high affinity nicotine binding sites in the brain contain combinations of just the α4 and β2 subunits. Co-injections of RNA for these two subunits into Xenopus oocytes readily result in the expression of receptors with pharmacological properties consistent with those of the brain's high affinity receptors (Nelson et al., 2003). Likewise, co-expression of α3 and β4 subunits, strongly expressed in the peripheral nervous system, yields receptors used to model ganglionic receptors. All pairwise combinations of α2, α3, or α4 with either β2 or β4 are easily characterized in the oocyte expression systems, and those results can be extended to model more complex subunit combinations through the analysis of the effects of systematic addition of other subunits (Gerzanich et al., 1998, Papke et al., 2007).

The first practical targeting of neuronal nicotinic acetylcholine receptors (nAChR) for a therapeutic indication came with the development of ganglionic blockers, such as hexamethonium and mecamylamine, for the treatment of hypertension (Stone et al., 1956). While the use of ganglionic blockers for the treatment of hypertension has been eclipsed by the subsequent development of more effective antihypertensive agents with better side-effect profiles, there is a renewed interest in antagonists that may inhibit nAChRs in brain (Dwoskin et al., 2004, Rose et al., 1994a, Rose et al., 1994b). Of necessity, for human studies this renewed interest has focused on the classical, and largely nonselective blocker, mecamylamine, since it is the only CNS active nAChR antagonist approved for use in humans. Mecamylamine has been shown to have usefulness as an adjunct therapy in the treatment of Tourette's syndrome (Sanberg et al., 1998) and, in combination with nicotine replacement therapy, to improve clinical effectiveness in smoking cessation therapy (Rose et al., 1994b).

As with any course of therapeutic development, the diversity of receptor subtypes associated with different indications has created a need for the development of subtype-selective drugs. Unfortunately, nAChRs in brain show diversity and subtlety of function that, arguably, is unsurpassed by any other receptor system in the CNS. Several subtype-selective agonists and partial agonists are now in clinical trails for various indications (Olincy et al., 2006, Potts and Garwood, 2007, Wilens et al., 2006). However, the development of subtype-selective antagonists has proven to be a more daunting challenge. One reason for this is that some complex subunit combinations that may exist in vivo are not easily recreated in artificial expression systems. This is particularly true for receptor subtypes containing the α6 subunit (Dowell et al., 2003, Kuryatov et al., 2000). Nonetheless, because α6-containing receptors are likely involved in nicotine dependence (Azam et al., 2007, Azam and McIntosh, 2006), they have been proposed to be a potentially useful target for therapies using nicotinic receptor antagonists (Dwoskin et al., 2004, Zoli et al., 2002). We have extended the characterization of the commonly used nAChR antagonists mecamylamine and dihydro-β-erythroidine (DHβE), and herein report a new chimera that may be useful for identifying α6-selective noncompetitive antagonists. We utilize that chimera to test a series of bis-quaternary ammonium compounds, identifying structural rigidity of the analogs as a potentially important feature for selective inhibition of receptors containing the α6 intracellular and transmembrane domains.