Gene targeting demonstrates that α4 nicotinic acetylcholine receptor subunits contribute to expression of diverse [³H]epibatidine binding sites and components of biphasic ⁸⁶Rb⁺ efflux with high and low sensitivity to stimulation by acetylcholine

Abstract

[³H]Epibatidine binds to nAChR subtypes in mouse brain with higher (K_D ≈ 0.02 nM) and lower affinity (K_D ≈ 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of α7, β2 or β4 subunits. Most, but not all, higher and lower affinity binding sites require β2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the α7, β2 or β4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [³H]epibatidine. Mol. Pharmacol. 70, 947–959). Effects of functional α4 gene deletion are reported here. Deletion of α4 virtually eliminated cytisine-sensitive, higher-affinity [³H]epibatidine binding as did β2 deletion, confirming that these sites are α4β2*-nAChR. Cytisine-resistant, higher-affinity [³H]epibatidine binding sites are diverse and some of these sites require α4 expression. Lower affinity [³H]epibatidine binding sites are also heterogeneous and can be subdivided into α-bungarotoxin-sensitive and -resistant components. Deleting α4 did not affect the α-bungarotoxin-sensitive component, but markedly reduced the α-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of β2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of α4 subunits. The effects of α4 gene targeting on receptor function were measured using a ⁸⁶Rb⁺ efflux assay. Concentration–effect curves for ACh-stimulated ⁸⁶Rb⁺ efflux are biphasic (EC₅₀ values = 3.3 μM and 300 μM). Targeting α4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of β2. Thus, α4β2*-nAChRs mediate a significant fraction of both phases of ACh stimulated ⁸⁶Rb⁺ efflux.

Introduction

Nicotinic cholinergic receptors (nAChRs) are ligand-gated ion channels expressed throughout the body, particularly in skeletal muscle, autonomic ganglia, and the central nervous system. Neuronal nAChRs are pentameric assemblies of homologous subunits, 11 of which have been identified (α2–α7, α9, α10, β2–β4) (Lindstrom, 2000). nAChR influence simple and complex behaviors (Picciotto, 2003), regulate acute and chronic responses to nicotine and similar drugs (Dani and DeBiasi, 2001), and have been implicated in aspects of Alzheimer's and Parkinson's diseases as well as schizophrenia, Tourette's syndrome, and anxiety (Bourin et al., 2003, Leonard et al., 2001, Quik, 2004). Different nAChRs appear to regulate these behaviors and diseases making it important to identify subtype specific function. The potential for nAChR diversity is considerable, but is limited by rules of receptor assembly for and cellular expression of nAChR subunits (Millar, 2003). Nevertheless, overlapping expression of compatible subunits makes identifying the regional brain compositions of the nAChRs challenging.

Radioligand binding is very useful for the identification of neuronal nAChR subtypes. In particular, epibatidine is an extraordinarily potent nicotinic agonist (Badio and Daly, 1994) that binds to many, if not all, nAChR subtypes in brain (Houghtling et al., 1995, Marks et al., 1998, Marks et al., 2006, Zoli et al., 1998, Perry et al., 2002) and to those heterologously expressed in Xenopus oocytes (Parker et al., 1998; Kuryatov et al., 2000) or HEK cells (Xiao and Kellar, 2004). Immunochemical (Whiting and Lindstrom, 1988, Flores et al., 1992, Gotti et al., 2005a, Gotti et al., 2005b, Marritt et al., 2005) or gene deletion (Picciotto et al., 1995, Xu et al., 1999, Zoli et al., 1998, Orr-Urtreger et al., 1997, Marubio et al., 1999, Ross et al., 2000, Whiteaker et al., 2002, Marks et al., 2006) methods confirm the existence of multiple epibatidine binding subtypes.

[³H]Epibatidine binding can be separated into two major classes that differ markedly in affinity (K_D ≈ 0.02 nM and K_D ≈ 5 nM) (Marks et al., 1999, Whiteaker et al., 2000b). Densities of higher- and lower affinity [³H]epibatidine binding sites in rodent brain are approximately equal. Each of these two classes of binding sites can be subdivided on the basis of differential sensitivity to inhibition by nicotinic agonists and antagonists. Higher affinity [³H]epibatidine binding sites can be separated into cytisine-sensitive and -resistant components. Lower affinity [³H]epibatidine binding sites can be separated into α-bungarotoxin-sensitive and -resistant components (Marks et al., 1998, Whiteaker et al., 2000a, Perry et al., 2002). Recently, Marks et al. (2006) reported that deletion of either β2 or β4 markedly reduced many components of both higher and lower affinity [³H]epibatidine binding sites whereas α7 deletion eliminated only the α-bungarotoxin sensitive component of lower affinity [³H]epibatidine binding. Clearly, an α subunit is required to form functional nAChRs. The studies reported here evaluated the effects of α4 gene deletion on diverse [³H]epibatidine binding sites and indicate that α4 is essential to the expression of many [³H]epibatidine binding sites.

Characterization of binding sites provides significant information about nAChR diversity, but characterization of nAChR function provides significant additional information. Electrophysiological methods have been successfully used to demonstrate functional diversity (Alkondon and Albuquerque, 1995) and examine changes in expression following nAChR subunit deletion (Picciotto et al., 1995, Orr-Urtreger et al., 1997, Marubio et al., 1999). The function of nAChRs has also been measured using biochemical methods. Many nAChRs are expressed on presynaptic nerve terminals, and the function of these receptors has been evaluated by measuring neurotransmitter release from synaptosomes or tissue slices (Wonnacott, 1997). Agonist-stimulated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes provides a direct biochemical assay for nAChR function (Marks et al., 1994, Marks et al., 1999, Marks et al., 2002). Efflux with pharmacological properties consistent with an α3β4-nAChR is seen in a few brain regions (inferior colliculus and interpeduncular nucleus) (Marks et al., 2002), but β2* nAChRs modulate this response in most brain regions (Marks et al., 1999, Marks et al., 2000). Biphasic agonist dose–response curves have been described for α4β2-nAChR expressed in cell lines or Xenopus oocytes (Zwart and Vijverberg, 1998, Buisson and Bertrand, 2001, Nelson et al., 2003, Zhou et al., 2003). The observation that concentration–effect curves for acetylcholine (Ach) stimulation of agonist-stimulated ⁸⁶Rb⁺ efflux are also biphasic suggests that α4β2* nAChRs modulate both of these components of agonist-stimulated ⁸⁶Rb⁺ efflux.

The studies reported here evaluated the effects of α4 gene deletion on multiple components of [³H]epibatidine binding as well as ACh-stimulated ⁸⁶Rb⁺ efflux. Major findings include the demonstration of a previously undescribed low affinity a4β2* nAChR and confirmation that α4β2* nAChRs modulate both the high and low affinity components of agonist-stimulated ⁸⁶Rb⁺ efflux. Portions of this work have been published as an abstract (Marks et al., 2003).