Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors
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.
Section snippets
Materials
[3H]MLA was synthesised in collaboration with Tocris Cookson (Bristol, UK). This was prepared essentially as described for the production of deuteriated methylsuccinic acid by Hardick et al. (1996b) and fully characterised (Blagbrough et al., 1994, Coates et al., 1995). Different batches of radioligand ranged in specific activity from 25-50 Ci/mmol. [3H]epibatidine (specific radioactivity 33.8 Ci/mmol) was obtained from NEN Dupont (Boston, MA) and [125I]NaI was obtained from Amersham
Optimisation of [3H]MLA binding conditions
In initial studies to establish the conditions for [3H]MLA binding to brain tissue, phosphate and HEPES buffers (pH 7.4) were compared, with and without various combinations of Tris, EDTA and Ringer salts (118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2). In the absence of tissue, there was some displaceable binding to glass fibre filters, but this was eradicated by addition of 0.1% (w/v) BSA to the incubation buffer. BSA was subsequently always included in [3H]MLA binding assays. Incubation of rat brain
Discussion
[3H]MLA binds to rat brain membranes with an affinity, pharmacological profile and regional distribution characteristic of αBgt-sensitive, putative α7 subunit-containing nAChRs. This is consistent with the known properties and specificity of MLA and related norditerpenoid plant alkaloids (MacAllan et al., 1988, Ward et al., 1990, Wonnacott et al., 1993). The saturation binding data (Fig. 2) show that [3H]MLA binds with high affinity (Kd=1.86 nM) to an apparently homogeneous population of
Acknowledgements
This study was supported by The Wellcome Trust, Grant Nos 036214 and 045023. We gratefully acknowledge Adrian Mogg for the provision of rat muscle receptor preparations.
References (55)
- et al.
Homomeric and native α7 acetylcholine receptors exhibit remarkably similar but non-identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex
FEBS Lett.
(1993) - et al.
Acylation of lycoctonine: semi-synthesis of inuline, delsemine analogues and methyllycaconitine
Tetrahedron Lett.
(1994) - et al.
A rapid filtration assay for soluble receptors using polyethylimine-treated filters
Anal. Biochem.
(1983) - et al.
The α-bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the α7 subunit
J. Biol. Chem.
(1997) The fall and rise of neuronal α-bungarotoxin binding proteins
Trends Pharmacol. Sci.
(1992)- et al.
Rapid and efficient isolation of the nicotinic receptor antagonist methyllycaconitine from delphinium—Assignment of the methylsuccinimide absolute stereochemistry as S
Tetrahedron Lett.
(1994) - et al.
An HPLC assay for the norditerpenoid alkaloid methyllycaconitine, a potent nicotinic acetylcholine receptor antagonist
J. Pharm. Biomed. Anal.
(1995) - et al.
Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions
J. Biol. Chem.
(1995) - et al.
Methyllycaconitine: a novel nicotinic antagonist
Mol. Cell Neurosci.
(1992) - et al.
The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit β4
Neuron
(1989)
α9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells
Cell
A functional α-bungarotoxin receptor is present in chick cerebellum: purification and characterisation
Neuroscience
Conversion of the sodium channel activator aconitine into a potent α7-selective nicotinic ligand
FEBS Lett.
Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems
FEBS Lett.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples
Anal. Biochem.
Nicotinic receptors in the development and modulation of CNS synapses
Neuron
Nicotinic acetylcholine receptor from rat brain: solubilization, partial purification and characterization
J. Biol. Chem.
Distribution of α-bungarotoxin binding sites in the central nervous system and peripheral organs of the rat
Toxicon
Brain α-bungarotoxin-binding protein cDNAs and mABs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily
Neuron
Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain
Brain Res.
Assembly of human neuronal nicotinic receptor α5 subunits with β3, β2 and β4 subunits
J. Biol. Chem.
Methyllycaconitine: a selective probe for neuronal β-bungarotoxin binding site
FEBS Lett.
Neuronal nicotinic acetylcholine receptor β-subunit is coded for by the cDNA clone α4
FEBS Lett.
Interrelationship of carbohydrate and the α-toxin binding site on the acetylcholine receptor from Torpedo Marmorata
Life Sci.
Nicotinic acetylcholine receptors in separate brain regions exhibit different affinities for methyllycaconitine
Neuroscience
Blockade of nicotinic currents in hippocampal-neurons defines methyllycaconitine as a potent and specific receptor antagonist
Mol. Pharmacol.
Choline is a selective agonist of α7 nicotinic acetylcholine receptors in the rat brain neurons
Eur. J. Neurosci.
Cited by (238)
Norditerpenoid alkaloids from Aconitum and Delphinium: structural relevance in medicine, toxicology, and metabolism
2021, Natural Product ReportsStructural Studies of Norditerpenoid Alkaloids: Conformation Analysis in Crystal and in Solution States
2021, European Journal of Organic ChemistryStimulation of brain α7-nicotinic acetylcholine receptors suppresses the rat micturition through brain GABAergic receptors
2021, Biochemical and Biophysical Research CommunicationsNicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation
2020, International Journal for Parasitology: Drugs and Drug Resistance