Amino Acid Residues That Confer High Selectivity of the 6 Nicotinic Acetylcholine Receptor Subunit to -Conotoxin MII[S4A,E11A,L15A] (original) (raw)
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Analogs of -Conotoxin MII Are Selective for 6-Containing Nicotinic Acetylcholine Receptors
Molecular Pharmacology, 2004
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. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC 50 at ␣64 versus ␣34 nAChRs. The peptide had a 590-fold lower IC 50 for ␣6/␣32 versus ␣32 and a 2020-fold lower IC 50 for ␣6/␣323 versus ␣32 nAChRs. MII[H9A;L15A] had little or no activity at ␣22, ␣24, ␣34, ␣42, ␣44, and ␣7 nAChRs. Functional block by MII[H9A;L15A] of rat ␣6/␣323 nAChRs (IC 50 ϭ 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [ 125 I]␣-conotoxin MII binding to putative ␣62* 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.
1999
The ␣-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication. In this study, the native ␣-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the AChinduced current in all rat parasympathetic neurons examined, with IC 50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified ␣-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC 50 value of 375 nM and a maximally effective concentration caused 91% block. [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in ϳ80% of neurons, with an IC 50 value of 1.4 nM and 46% maximal block of the total current. The residual current was not inhibited further by ␣-bungarotoxin, but was further reduced by the ␣-conotoxins PnIA or PnIB, and by mecamylamine.
Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors
Acta Pharmacologica Sinica, 2009
Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.
Journal of Biological Chemistry, 2018
Edited by Wolfgang Peti Nicotinic acetylcholine receptors (nAChRs) containing ␣6 and 4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate ␣-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat ␣6/␣34 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to ␣-conotoxins. Sequence homology comparisons of human and rat ␣64 nAChR subunits indicated that ␣6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of 4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of ␣-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro 13 in close proximity to residue 119 of the AChBP complementary subunit. Sitedirected mutagenesis studies revealed that Leu 119 of human 4 contributes to higher sensitivity of human ␣6/␣34 nAChRs to ␣-conotoxins, and structure-activity studies indicated that PeIA Pro 13 is critical for high potency. Human and rat ␣6/␣34 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro 13 and residue 119 of the 4 subunit. These results highlight the potential significance of species differences in ␣64 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.
Biochemistry, 1999
The mammalian skeletal muscle acetylcholine receptor contains two nonequivalent acetylcholine binding sites, one each at the R/δ and R/γ subunit interfaces. R-Conotoxin MI, a 14-amino acid competitive antagonist, binds at both interfaces but has ∼10 4 higher affinity for the R/δ site. We performed an "alanine walk" to identify the residues in R-MI that contribute to this selective interaction with the R/δ site. Electrophysiological measurements with Xenopus oocytes expressing normal receptors or receptors lacking either the γ or δ subunit were made to assay toxin-receptor interaction. Alanine substitutions in most amino acid positions had only modest effects on toxin potency at either binding site. However, substitutions in two positions, proline-6 and tyrosine-12, dramatically reduced toxin potency at the high-affinity R/δ site while having comparatively little effect on low-affinity R/γ binding. When tyrosine-12 was replaced by alanine, the toxin's selectivity for the high-affinity site (relative to that for the low-affinity site) was reduced from 45 000-to 30-fold. A series of additional amino acid substitutions in this position showed that increasing side chain size/hydrophobicity increases toxin potency at the R/δ site without affecting R/γ binding. In contrast, when tyrosine-12 is diiodinated, toxin binding is nearly irreversible at the R/δ site but also increases by ∼500-fold at the R/γ site. The effects of position 12 substitutions are accounted for almost entirely by changes in the rate of toxin dissociation from the high-affinity R/δ binding site.
alpha.-Conotoxin EI, A New Nicotinic Acetylcholine Receptor Antagonist with Novel Selectivity
Biochemistry, 1995
We report the isolation and characterization of a novel nicotinic acetylcholine receptor (nAChR) ligand. The toxin is an 18 amino acid peptide and is the first reported a-conotoxin from an Atlantic fish-hunting Conus. The peptide was purified from the venom of Conus emineus and is called a-conotoxin EI. The sequence diverges from that of previously isolated a-conotoxins. We demonstrate that this structural divergence has functional consequences. In Torpedo nAChRs, a-conotoxin EI selectively binds the agonist site near the a/d subunit interface in contrast to a-conotoxin MI which selectively targets the a / y agonist binding site. In mammalian nAChRs a-conotoxin EI shows high affinity for both the a/d and a / y subunit interfaces (with some preference for the a/d site), whereas a-conotoxin MI is highly selective for the d d ligand binding site. The sequence of the peptide is: Arg-Asp-Hyp-Cys-Cys-Tyr-His-Pro-Thr-Cys-Asn-Met-Ser-Asn-Pro-Gln-Ile-Cys-NH~, with disulfide bridging between Cys4-CyslO and CysS-Cysl8, analogous to those of previously described a-conotoxins. This sequence has been verified by total chemical synthesis. Thus, a-conotoxin EI is a newly-available tool with unique structure and function for characterization of nAChRs. Nicotinic acetylcholine receptors (nAChRs)' in skeletal muscle and the electric organ of Torpedo are heteropentameric ligand-gated cation channels formed by four subunits in the stoichiometry (al)& 76. Several small molecule toxins isolated from plants, coral, and gastropods as well as polypeptide toxins from predatory organisms have been isolated which target nAChRs [for review see Chiappinelli (1993)l. The availability of these toxins has played a critical role in the progressive understanding of the structure and function of the nicotinic receptor. The nAChR requires two molecules of acetylcholine to bind two separate sites for channel opening. These nonequivalent binding sites are located at the a l y and al6 subunit interfaces (Blount & Merlie, 1989). Curariform antagonists bind with 1-2 orders of magnitude higher affinity to the a l y site than to the al6 site of both mammalian muscle and Torpedo receptors + This work was supported by NIMH Scientist Development Award for Clinicians K20 MHO0929 (J.M.M.), NIH Grants GM 48677 (B.M.O.) and NS 29951 (S.N.A.), and the Smokeless Tobacco Research Council (S.N.A.).
FEBS Letters, 2003
The Xenopus laevis oocyte expression system was used to determine the activities of K K-conotoxins EpI and the ribbon isomer of AuIB, on de¢ned nicotinic acetylcholine receptors (nAChRs). In contrast to previous ¢ndings on intracardiac ganglion neurones, K K-EpI showed no signi¢cant activity on oocyte-expressed K K3L L4 and K K3L L2 nAChRs but blocked the K K7 nAChR with an IC 50 value of 30 nM. A similar IC 50 value (103 nM) was obtained on the K K7/5HT 3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed K K3L L4 receptors but unlike in native cells, where it was 10-fold more potent than native K K-AuIB, had 25-fold lower activity. These results indicate that as yet uniden-ti¢ed factors in£uence K K-conotoxin pharmacology at native versus oocyte-expressed nAChRs. ß
Differential Targeting of Nicotinic Acetylcholine Receptors by Novel αA-Conotoxins
Journal of Biological Chemistry, 1997
We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12-18 residue, 4 Cys-containing ␣-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named ␣A-conotoxins EIVA and EIVB, block both Torpedo and mouse ␣1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to ␣-bungarotoxin, ␣A-EIVA is inactive at ␣7-containing nAChRs even at micromolar concentrations. In this regard, ␣A-EIVA is similar to the previously described ␣-conotoxins (e.g. ␣-MI and ␣-GI) which also selectively target ␣1versus ␣7-containing nAChRs. However, ␣-MI and ␣-GI discriminate between the ␣/␦ versus ␣/␥ subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, ␣A-conotoxin EIVA blocks both the ␣/␥ site and ␣/␦ site with equally high affinity but with distinct kinetics. The ␣A-conotoxins thus represent novel probes for the ␣/␥ as well as the ␣/␦ binding sites of the nAChR.
Journal of Biological Chemistry, 1999
We have investigated the molecular determinants responsible for ␣-bungarotoxin (␣Bgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous ␣ subunits, one highly sensitive to ␣Bgtx block (␣1) and the other, ␣Bgtx-insensitive (␣3). By replacing rat ␣3 residues 184-191 with the corresponding region from the Torpedo ␣1 subunit, we introduced a cluster of five ␣1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the ␣3 subunit. Functional activity and ␣Bgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric ␣3 subunit (␣3/␣1[5]) with either rat 2 or 4 subunits. Agonist-evoked responses of ␣3/␣1[5]-containing receptors were blocked by ␣Bgtx with nanomolar affinity (IC 50 values: 41 nM for ␣3/␣1[5]2 and 19 nM for ␣3/␣1[5]4). Furthermore, receptors containing the single point mutation ␣3K189Y acquire significant sensitivity to ␣Bgtx block (IC 50 values: 186 nM for ␣3K189Y2 and 179 nM for ␣3K189Y4). Another ␣3 chimeric subunit, ␣3/␣7[6], similar to ␣3/␣1[5] but incorporating the corresponding residues from the ␣Bgtx-sensitive ␣7 subunit, also conferred potent ␣Bgtx sensitivity to chimeric receptors when co-expressed with the 4 subunit (IC 50 value ؍ 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the ␣Bgtx-sensitive subunits ␣1 and ␣7 play a critical functional role in the interaction of ␣Bgtx with nicotinic acetylcholine receptors sensitive to this toxin.