Characterisation of the binding of [³H]methyllycaconitine: a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

Abstract

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for αbungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [³H]MLA bound to rat brain membranes with high affinity (K_d=1.86±0.31 nM) with a good ratio of specific to non-specific binding. The binding of [³H]MLA was characterised by rapid association (t_{$\text{1}\text{2}$}=2.3 min) and dissociation (t_{$\text{1}\text{2}$}=12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with αbungarotoxin-sensitive nAChR. The snake α-toxins, αbungarotoxin and αcobratoxin, displaced [³H]MLA with high affinity (K_i=1.8±0.5 and 5.5±0.9 nM, respectively), whereas nicotine was less potent (K_i=6.1±1.1 μM). The distribution of [³H]MLA binding sites in crudely dissected rat brain regions was identical to that of [¹²⁵I]αbungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [³H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake αtoxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [³H]MLA, therefore, is a novel radiolabel for characterising α7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled αbungarotoxin for rapid and accurate equilibrium binding assays.

Introduction

The snake toxin αbungarotoxin (αBgt) has been an invaluable tool in the characterisation of nicotinic acetylcholine receptors (nAChR) in skeletal muscle, for functional studies, as a pseudo-irreversible antagonist, and by labelling nAChR for quantitating, purifying and visualising receptors. With regard to nAChR in neurones, αBgt has had a chequered history: neuronal nAChR appeared to be insensitive to the toxin despite its ability to label specific binding sites which displayed a nicotinic pharmacology (reviewed by Clarke, 1992). The advent of tritiated nicotinic agonists (nicotine, ACh, cytisine and methylcarbamylcholine) in the 1980’s led to the characterisation of high (nM) affinity nicotinic agonist binding sites in vertebrate brain that were distinct, in pharmacology and distribution, from αBgt binding sites that displayed low (μM) affinity for agonists (Clarke et al., 1985, Marks et al., 1986, Wonnacott, 1986). Controversy surrounded the biological significance of neuronal αBgt binding sites: the large size of the toxin (and consequent concerns over its ability to access synaptic receptors) and its slow onset of binding and inhibition added to the debate. Eventually the cloning and expression of the α7 nAChR subunit (Couturier et al., 1990, Schoepfer et al., 1990, Seguela et al., 1993) legitimised the claim that αBgt binding proteins are indeed nAChR, homologous in structure and function to their muscle counterpart. The high relative Ca²⁺ permeability, and temporal and regional expression patterns of α7-type nAChR, have since made it the focus of considerable research efforts (reviewed by Role and Berg, 1996).

Molecular cloning of neuronal nAChR subunits has revolutionised our perspective of these receptors. Whereas the muscle nAChR is a heteropentamer, comprised of two copies of an α (agonist-binding) subunit and one each of β, γ and δ subunits (with a developmental switch from γ to ε), eight α-like subunits (α2-α9) and three non-α subunits (β2-β4) have so far been cloned from, and shown to be expressed in, neurones (Lindstrom et al., 1995). The minimum subunit combinations capable of creating functional nAChR on expression in Xenopus oocytes have constrained views of the subunit composition of native neuronal nAChR. Thus pairwise combination in Xenopus oocytes of α2, α3 or α4 with β2 or β4 generates functional receptors (Boulter et al., 1987, Duvoisin et al., 1989), but to date experimental evidence only substantiates the α4β2 combination as a native nAChR, which accounts for about 90% of high affinity tritiated agonist binding sites in the brain (Whiting et al., 1987, Flores et al., 1992). Given that the α5, α6 and β3 subunits are apparently incapable of forming nAChR when paired with any other subunit from the same species in Xenopus oocytes, together with the precedent for multiple types of subunit within the prototype muscle nAChR, more complex subunit combinations are plausible and evidence for neuronal nAChR comprised of combinations of three or four different subunits is accruing (Conroy and Berg, 1995, Ramirez-Latorre et al., 1996, Wang et al., 1996, Forsayeth and Kobrin, 1997).

The α7, α8 and α9 subunits are unique among α subunits for their capacity to form robust, homomeric nAChR that are sensitive to αBgt when expressed in Xenopus oocytes. This has generated intense debate about the nature of native nAChR containing these subunits. Although α7 and α8 subunits co-assemble in chicken nAChR (Keyser et al., 1993, Gotti et al., 1994), the apparent absence of α8 in mammals, and the restricted localisation of α9 (Elgoyhen et al., 1994), has focussed attention on the α7 subunit in mammalian neurones. The hypothesis that the major αBgt-sensitive neuronal nAChR in rat brain is a homomer of α7 subunits is advocated by the concordance between brain [¹²⁵I]αBgt binding sites and presumably homomeric α7 nAChR expressed in a pituitary cell line (Quik et al., 1996), and by the failure to detect any other known subunits in nAChR purified from rat brain using αBgt as an affinity ligand and probed by Western blotting with antibodies to known nAChR subunits (Chen and Patrick, 1997). However, other lines of evidence suggest that native α7-containing nAChRs are more complex than oocyte expression studies indicate. A number of unidentified proteins co-purify with the α7 subunit when αBgt-sensitive nAChR are purified from chick cerebellum (Gotti et al., 1992). α7 subunits are notoriously difficult to express in non-neuronal mammalian cell lines (Cooper and Millar, 1997), suggesting some neurone-specific components are required to form functional receptors. It has also been noted that, although similar, some differences do exist between native αBgt binding sites and α7 homomers expressed in Xenopus oocytes (Anand et al., 1993).

To explore the heterogeneity of neuronal nAChR, more selective, potent pharmacological tools are needed. Methyllycaconitine (MLA) is a natural norditerpenoid alkaloid, identified as the principal toxic agent in Delphinium brownii (Nambi-Aiyar et al., 1979) and present in other Delphinium and Consolida species (Wonnacott et al., 1993). MLA inhibits [¹²⁵I]αBgt binding to vertebrate brain with an affinity in the low nanomolar range, and has at least 100-fold selectivity for this site compared with other nicotinic ligand binding sites, including muscle (MacAllan et al., 1988, Ward et al., 1990, Wonnacott et al., 1993). MLA acts as a reversible, competitive antagonist, and picomolar concentrations are sufficient to inhibit homomeric α7 nAChR in Xenopus oocytes (Palma et al., 1996) and native α7-like nAChR in cultured hippocampal neurones (Alkondon et al., 1992). Concentrations of 100 nM or greater are required for substantial inhibition of α3β2 nAChR (Drasdo et al., 1992) whereas α4β2 and muscle nAChR require micromolar concentrations of MLA for blockade (Drasdo et al., 1992, Tian et al., 1997).

Structure-activity relationship studies have identified the 2-(methylsuccinimido) benzoyl moiety as critical for the nicotinic potency of MLA, although selectivity for α7-type nAChR appears to reside in the norditerpenoid core (Hardick et al., 1995, Hardick et al., 1996a). These studies have developed the chemistry for manipulating the MLA structure. This has been exploited for the incorporation of radioisotopes: a tritiated version of MLA would have several advantages over [¹²⁵I]αBgt, including rapid determination of true equilibrium binding constants and ease of handling. Deuteriation of MLA, via synthesis of deuteriated methylsuccinic acid (Hardick et al., 1996b), gave a product indistinguishable from natural MLA in its binding potency (Hardick et al., submitted) and this methodology has now been used to generate a tritiated version (Fig. 1). We describe here the initial characterisation of [³H]MLA, a novel radioligand for the study of α7-type nAChRs.