Analogs of α-Conotoxin MII Are Selective for α6-Containing Nicotinic Acetylcholine Receptors

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. ␣-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related ␣3 and ␣6 subunits. Structure-function analysis of ␣-conotoxin MII was performed in an attempt to generate analogs with preference for ␣6-containing [␣6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at ␣3* versus ␣6* nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. 125 I]␣-conotoxin MII binding to putative ␣6␤2* nAChRs in mouse brain homogenates (K i ϭ 3.3 nM). Thus, structure-function analysis of ␣-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing ␣3 and ␣6 subunits.nAChRs activated by the endogenous neurotransmitter acetylcholine belong to the superfamily of ligand-gated ion channels that also includes GABA A , 5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). These different ligand-gated ion channels show considerable sequence and structural homology. Each of the subunits has a relatively hydrophilic amino terminal half (ϳ200 amino acids) that constitutes an extracellular domain. This is followed by three hydrophobic transmembrane domains, a large intracellular loop, and then a fourth hydrophobic transmembrane span.A large number of genes have been cloned that encode subunits of nAChRs. It has been proposed that these subunits may be divided into subfamilies on the basis of both gene structure and mature protein sequence. The subunits ␣2, ␣3, ␣4, and ␣6 belong to subfamily III, tribe 1; ␤2 and ␤4 belong to tribe III-2; and the putative structural subunits ␣5 and ␤3 belong to tribe III-3 (Corringer et al., 2000). Within tribe III-1, subunits ␣3 and ␣6 show considerable sequence identity (ϳ80% in the ligand-binding extracellular domain). Thus, designing ligands to distinguish between ␣3* 1 and ␣6* is particularly challenging.␣-Conotoxin MII is a 16 amino acid peptide originally isolated from the venom of the marine snail Conus magus. This peptide potently targets neuronal in preference to the muscle subtype of nicotinic receptor with high affinity for both ␣3␤2 and ␣6* nAChRs. Unfortunately, ␣-conotoxin MII may not distinguish well between ␣3* and ␣6* nAChRs (Kuryatov et al., 2000). In an effort to remedy this situation and produce a selective ligand for ␣6* nAChRs, we have generated a series of ␣-conotoxin MII analogs.The ␣6 subunit is expressed in catecholaminergic neurons and in retina (Le Novère et al., 1996Vailati et al., 1999). In striatum, ␣6* nAChRs seem to play a central role in the modulation ...

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

“…As mentioned in the Introduction, ␣6 NTD /␣3 TMD/ICL and ␣6 NTD /␣4 TMD/ICL subunits form functional receptors together with ␤ subunits in heterologous expression systems (22)(23)(24)(25)(26)(27). In contrast, co-expression of ␣3 NTD /␣6 TMD/ICL and ␣4 NTD / ␣6 TMD/ICL chimeras with ␤2 or ␤2␤3 in oocytes does not result in functional receptors, and although the chimeras have been reported to form some functional receptors with ␤4, these are characterized by dramatically impaired functionalities compared with WT ␣3␤4 and ␣4␤4 nAChRs (23,27).…”

“…Several approaches have been applied to overcome this obstacle. First, co-expression of chimeric ␣6/␣3 or ␣6/␣4 subunits (␣6-NTD fused with ␣3-or ␣4-TMD/ICL) with ␤2, ␤2␤3, and ␤4 subunits results in formation of functional receptors in both mammalian cells and oocytes (22)(23)(24)(25)(26)(27). Second, the minute responses observed for ␣6␤2␤3 and ␣6␤4␤3 nAChRs in oocytes have been found to be dramatically enhanced by the introduction of a ␤3 V273S mutant in the receptors (28,29).…”

Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine- and Cocaine-Conditioned Reward in Mice

“…It is an analog of aconotoxin MII that has a high selectivity for a6* nAChRs. For example, a-conotoxin MII [H9A;L15A] has up to a 2020-fold selectivity for a6* vs a3*, with little or no activity at other nAChRs (a2b2, a2b4, a3b4, a4b2, a4b4, and a7) (McIntosh et al, 2004). a-Conotoxin MII [H9A;L15A] was dissolved in small aliquots of saline and stored in an À 18 to À 20 1C freezer until use.…”

“…The toxin was administered to each animal centrally at the following sites: lateral ventricles, nucleus accumbens, or septum. The doses used in our studies were calculated based on affinity and the potency of the compound at a6*-nAChRs (McIntosh et al, 2004).…”

Nicotinic receptors as CNS targets for Parkinson's disease

“…In a continued effort to more precisely determine the subunit composition of these receptors, experiments were recently performed with α-conotoxinMII analogs to determine whether these might discriminate between α6β2* subtypes. One such analog α-conotoxinMII E11A, in which the glutamic acid at position 11 is replaced with alanine [113], inhibited 125 I-α-conotoxinMII binding to striatum from mice, monkeys and humans in a biphasic manner (Fig. 2) [88].…”