The subunit composition and pharmacology of α-Conotoxin MII-binding nicotinic acetylcholine receptors studied by a novel membrane-binding assay
Introduction
Nicotinic acetylcholine receptors (nAChR) comprise a diverse family of ligand-gated ion channels. Twelve genes that encode nAChR subunits (α2–α10 and β2–β4) have been identified in the autonomic and central nervous systems of vertebrates (reviewed in Lindstrom, 2003). Neuronal nAChR, like the nAChR expressed in skeletal muscle and in electric organs of eels and rays, are likely to be pentameric assemblies of homologous subunits that form a ligand-gated ion channel for which acetylcholine is the endogenous agonist (Lindstrom, 2003). Functional receptors may be formed by assembly of five identical subunits (most notably with α7-nAChR) or, more commonly, by the assembly of two or more different subunits (at least one α and one β). Even with restrictions on assembly dictated by subunit distribution and properties, the potential for diversity of nAChR subtypes is very high. Inasmuch as subunit composition influences virtually every aspect of receptor function including pharmacological properties, ion selectivity, duration of ion channel opening, the rates of activation and desensitization (Itier and Bertrand, 2001, Quick and Lester, 2002), identification of the subunit composition and anatomical distribution of those nAChR subtypes that are expressed naturally is essential for understanding the function of this receptor family.
Ligand binding assays are powerful tools that facilitate the study of naturally expressed nAChR. α-Bungarotoxin, originally identified as an antagonist of muscle-type nAChR, has been extensively used to study the properties of α7*, α8* and α9* nAChR (Seguela et al., 1992, Gotti et al., 1997). Nicotinic agonists have been valuable in the identification and characterization of a high affinity binding site identified as predominantly α4β2-nAChR using immunochemical methods and gene deletion strategies (Whiting and Lindstrom, 1987, Flores et al., 1992, Picciotto et al., 1995, Marubio et al., 1999, Ross et al., 2000). The relative selectivity of α-bungarotoxin and nicotine-like drugs for specific nAChR subtypes has restricted their utility in the identification of additional nAChR. This limitation has been alleviated by the isolation, characterization and radiolabelling of epibatidine, a very potent nicotinic agonist, that binds with high affinity to many different nAChR (Badio and Daly, 1994, Houghtling et al., 1995). Distinct subsets of epibatidine binding sites, which include those also labeled by α-bungarotoxin and nicotine, have been identified by selective inhibition of epibatidine binding by nicotinic agonists and antagonists (Marks et al., 1998, Parker et al., 1998, Whiteaker et al., 2000b). These differential inhibition assays have been extraordinarily useful in identifying nAChR subtypes in addition to α4β2 nAChR and α7 nAChR. However, the differential inhibition approach is not well suited to detailed pharmacological analyses or to the investigation of receptor subtypes that represent small fractions of the total receptor pool that binds epibatidine. For these studies, alternative ligands that label fewer, preferably a single nAChR subtype, are more optimal. α-Conotoxin MII (α-CtxMII) may be such a ligand.
α-Conotoxins are small polypeptide nAChR antagonists, that are components of the venoms of predatory marine snails of the genus Conus (McIntosh et al., 1999). α-CtxMII is a 16 amino acid peptide originally isolated from the venom of Conus magus by its ability to selectively inhibit the function of α3β2 nAChRs expressed in Xenopus oocytes (Cartier et al., 1996). Later, it was shown that α-CtxMII itself cannot satisfactorily differentiate α3 and α6 nAChR subunits (e.g. McIntosh et al., 2004). The fact that α-CtxMII potently and selectively inhibits a subset of the release of dopamine in rodent striatum stimulated by nicotinic agonists identifies endogenous functional α-CtxMII sensitive nAChR (Grady et al., 1997, Kulak et al., 1997, Kaiser et al., 1998, Salminen et al., 2004). These native binding sites are α6-containing nAChR especially in dopaminergic areas (e.g. McIntosh et al., 2004).
In order to localize these nAChR, the binding of [125I]-α-CtxMII to mouse brain was examined autoradiographically (Whiteaker et al., 2000a). Consistent with the functional data, specific binding was observed in dopaminergic regions. Additional specific binding was detected in medial habenula, interpeduncular nucleus, superior colliculus, geniculate nuclei and oculomotor nerve (at higher levels than in the dopaminergic regions). Studies using null mutant mice showed that the α3 subunit does not participate in [125I]-α-CtxMII binding sites in dopaminergic areas (striatum, nucleus accumbens, olfactory tubercles) and superior colliculus (Whiteaker et al., 2002). Studies done with α6 (Champtiaux et al., 2002), β3 (Cui et al., 2003) and β2 (Grady et al., 2001) null mutant mice indicate these subunits are critical components of [125I]-α-CtxMII binding nAChR in the central nervous system. α4 gene deletion abolished half of the [125I]-α-CtxMII binding (Marubio et al., 2003). Thus, in dopaminergic areas [125I]-α-CtxMII binding detects α6* nAChR (either α4α6β2β3 or α6β2β3). Recently, it was established (Champtiaux et al., 2003, Salminen et al., 2004) that each of these α-CtxMII sensitive nAChR subtypes are functional, using the dopamine release assay from mouse synaptosomes.
While the autoradiographic method provides excellent information about anatomical distribution of binding sites, it is not well suited for detailed pharmacological evaluations. Membrane binding assays are useful for such studies but previous efforts to develop such a binding assay have been undermined by very high levels of non-specific binding and correspondingly poor signal to noise ratios. Therefore, the purposes of this study were to develop a workable [125I]-α-CtxMII filtration-binding assay that uses tissue homogenates, and apply it to investigate the pharmacology and subunit compositions of α-CtxMII-binding nAChR. In the studies reported here [125I]-α-CtxMII binding pharmacology was characterized using brain membranes prepared from the striatum, olfactory tubercles and superior colliculus of C57BL/6 mice. The effects of α4, α5, α7, β2, β3 and β4 subunit-null mutations on [125I]-α-CtxMII membrane binding were also studied, and the results were compared to those from α-CtxMII-sensitive dopamine release (Salminen et al., 2004).
Section snippets
Animals
All procedures were reviewed and approved by the Animal Care and Utilization Committee of the University of Colorado, Boulder. Male C57BL/6 mice (60–90 days of age) as well as all genotypes of the subunit-null mutant mice, bred at the Institute for Behavioral Genetics, University of Colorado (Boulder, CO, USA), were maintained on a 12-h light/12-h dark cycle (light on from 7 AM to 7 PM), at 22 °C, with free access to food (Teklad Rodent Diet, Harlan, Madison, WI) and water. All nAChR
Results
α-CtxMII partially inhibited [125I]-epibatidine binding in olfactory tubercles, striatum and superior colliculus. The maximum α-CtxMII-inhibitable binding in olfactory tubercles is 3.74 ± 0.53 fmol/mg protein, which is 24% of total epibatidine binding; in striatum it is 7.89 ± 0.68 fmol/mg protein, which is 29% of total binding and in superior colliculus it is 16.51 ± 0.18 fmol/mg protein, which is 39% of total binding (Fig. 1). KI-values of α-CtxMII inhibition were 0.19 ± 0.01 nM for olfactory tubercles,
Discussion
α-CtxMII-sensitive sites are a fraction (≤30%) of total nAChR binding sites in the brain regions that express them, making a direct binding approach much preferable to a subtractive approach (as used for instance by Whiteaker et al. (2000b) for β4* nAChR binding studies, or Salminen et al. (2004) for α-CtxMII-sensitive functional studies). Although much has been learned about nAChR that bind [125I]-α-CtxMII using autoradiographic methods (see Section 1), membrane binding approaches are much
Acknowledgements
This work was supported by National Institute on Drug Abuse (NIDA) grant DA12242 (to M.J.M., P.W., J.M.M.), National Institute on Alcohol Abuse and Alcoholism grant AA-011156 (to A.C.C.), National Institute of Mental Health grant MH53631 (to J.M.M.), Colorado Tobacco Research IDEA grant 3I-030 (to P.W.), Colorado Tobacco Research Grant 3F-034 (to O.S.) and by an Academy of Finland grant (to O.S.). Production of the null mutant mice was supported by animal resources grant DA015663 from NIDA (to
References (41)
- et al.
A new α-conotoxin which targets α3β2 nicotinic acetylcholine receptors
Journal of Biological Chemistry
(1996) - et al.
Neuronal nicotinic receptors: from protein structure to function
FEBS Letters
(2001) - et al.
Protein measurement with the Folin phenol reagent
Journal of Biological Chemistry
(1951) - et al.
Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons
Journal of Comparative Neurology
(2002) - et al.
Effect of novel α-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes
Journal of Pharmacology and Experimental Therapeutics
(2004) - et al.
Epibatidine: a potent analgetic and nicotinic agonist
Molecular Pharmacology
(1994) - et al.
Distribution and pharmacology of α6-containing nicotinic acetylcholine receptors analyzed with mutant mice
Journal of Neuroscience
(2002) - et al.
Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice
Journal of Neuroscience
(2003) - et al.
Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction
Biochemistry and Pharmacology
(1973) - et al.
The β3 nicotinic receptor subunit: a component of α-conotoxin MII binding nAChRs which modulate dopamine release and related behaviors
Journal of Neuroscience
(2003)
Determinants of potency on α-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors
Biochemistry
A subtype of nicotinic cholinergic receptor in rat brain is composed of alpha4 and beta2 subunits and its upregulated by chronic nicotine treatment
Molecular Pharmacology
Alpha7, alpha8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties
European Journal of Neuroscience
Pharmacological comparison of transient and persistent [3H]dopamine release from mouse striatal synaptosomes and response to chronic l-nicotine treatment
Journal of Pharmacology and Experimental Therapeutics
Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release in striatum
Journal of Neurochemistry
Characterization of [3H]epibatidine binding to nicotinic receptors in rat and human brain
Molecular Pharmacology
Differential inhibition by a α-conotoxin MII of the nicotinic stimulation of [3H]dopamine release from rat striatal synaptosomes and slices
Journal of Neurochemistry
A study of the “desensitization” produced by acetylcholine at the motor end-plate
Journal of Physiology (London)
Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei
Journal of Neuroscience
α-Conotoxin MII blocks nicotine-stimulated dopamine release in rat striatal synaptosomes
Journal of Neuroscience
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