The .alpha.-Conotoxins GI and MI Distinguish between the Nicotinic Acetylcholine Receptor Agonist Sites while SI Does Not

Hann, Richard M.; Pagán, Oné R.; Eterović, Vesna A.

doi:10.1021/bi00251a014

Cited by 68 publications

(91 citation statements)

References 34 publications

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“…Our binding data reveal affinities of Ctx for the Torpedo ␣␥ site that differ from those observed by others (10,11). Some of the larger differences can be accounted for by the distinct buffer conditions used as follows: Hann et al (13) and Groebe et al (10) used low ionic strength buffers to measure binding in the presence of detergents.…”

Section: Table III Charge Effects On Binding Observed In Low Ionic Stcontrasting

confidence: 54%

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

Papineni

Sanchez

Baksi

et al. 2001

Journal of Biological Chemistry

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We have tested the importance of charge interactions for ␣-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on ␣-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the ␣␦ site but not at the ␣␥ site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the ␣␦ site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, ␣-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

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Section: Table III Charge Effects On Binding Observed In Low Ionic Stcontrasting

confidence: 54%

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

Papineni

Sanchez

Baksi

et al. 2001

Journal of Biological Chemistry

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“…Two ACh binding sites are formed between its a1/d and a1/g (or « in adult) interfaces. Sine et al (1995) demonstrated that a paralytic peptide, a-CTx MI, selectively binds the a1/d interface (Sine et al, 1995;Sugiyama et al, 1998); subsequently, other toxins were discovered that selectively bind the a1/d, a1/«, or a1/g interfaces (Hann et al, 1994;Groebe et al, 1995;Martinez et al, 1995;Molles et al, 2002a,b), which allowed the development of radiolabeled and fluorescent probes for specific labeling of fetal or adult muscle nAChR subtypes (Teichert et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Molecular Interaction of α-Conotoxin RgIA with the Rat α9α10 Nicotinic Acetylcholine Receptor

et al. 2015

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The a9a10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that a-conotoxin (a-CTx) RgIA, one of the few a9a10 selective ligands identified to date, is 300-fold less potent on human versus rat a9a10 nAChR. This species difference was conferred by only one residue in the (2), rather than (1), binding region of the a9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with a-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the a9(1) face and two residues on the a10(2) face of the a9a10 nAChR. However, mutations of these residues had little or no effect on toxin block of the a9a10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (a10 Ε197, P200 and a9 T61, D121) resulted in 19-to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human a9 nAChR, we modeled the rat a9a10 ECD and its complexes with a-CTx RgIA and acetylcholine. Our data support the interaction of a-CTx RgIA at the a10/a9 rather than the a9/a10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.

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“…A well defined subgroup of ␣-conotoxins, referred to as the ␣3/5 2 subfamily conotoxins (Table I), are antagonists of neuromuscular receptors and show, except for ␣-conotoxin SI (9,10), binding preference to the ␣ 1 /␥ subunit interface of Torpedo nAChR. On the other hand, more recently found ␣A-conotoxins differ from the ␣3/5 subfamily conotoxins in the amino acid sequences (11,12) as well as in their threedimensional structures (13).…”

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confidence: 99%

Solution Conformation of α-Conotoxin EI, a Neuromuscular Toxin Specific for the α1/δ Subunit Interface of Torpedo Nicotinic Acetylcholine Receptor

Park¹,

Suk²,

Jacobsen³

et al. 2001

Journal of Biological Chemistry

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A high resolution structure of ␣-conotoxin EI has been determined by 1 H NMR spectroscopy and molecular modeling. ␣-Conotoxin EI has the same disulfide framework as ␣4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the ␣3/5 and ␣A conotoxins. The unique binding preference of ␣-conotoxin EI to the ␣ 1 /␦ subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various ␣-conotoxins possessing distinct receptor subtype specificities. Structural comparison of ␣-conotoxin EI with the ␥-subunit favoring ␣-conotoxin GI suggests that the Torpedo ␦-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven ␣-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is ϳ20 Å (height) ؋ 20 Å (width) ؋ 15 Å (thickness).The nicotinic acetylcholine receptors (nAChRs) 1 are a well studied family of ligand-gated ion channels comprising a diverse set of molecular subtypes (1). The best characterized is the receptor at the neuromuscular junction, with four different subunits in a pentameric array, i.e. (␣ 1 ) 2 ␤ 1␥ ␦. Less well understood and more diverse are the neuronal nAChRs that assemble in exogenous expression systems with a general composition of (␣ m ) 2 (␤ n ) 3 , where m ϭ 2-6 and n ϭ 2-4, or (␣ 7 ) 5 (2, 3). A large variety of ligands bind to such diverse nAChRs via unknown mechanisms. A better understanding of the ligandbinding mechanism would be possible if a suitable three-dimensional structure of nAChR were available. Although cryoelectron microscopic images of nAChR show the spatial arrangement of five subunits of the receptor and some aspects of the ligand-binding pockets (4, 5), they are as yet insufficient for describing ligand-receptor interactions in atomic detail. In this regard, the recently determined crystal structure of acetylcholine-binding protein is expected to provide useful insight into ligand-nAChR interactions (6).Small peptide toxins of Conus origin known as the ␣-and ␣A-conotoxins are highly useful tools for exploring ligandnAChR interactions (7,8). A well defined subgroup of ␣-conotoxins, referred to as the ␣3/5 2 subfamily conotoxins (Table I), are antagonists of neuromuscular receptors and show, except for ␣-conotoxin SI (9, 10), binding preference to the ␣ 1 /␥ subunit interface of Torpedo nAChR. On the other hand, more recently found ␣A-conotoxins differ from the ␣3/5 subfamily conotoxins in the amino acid sequences (11, 12) as well as in their threedimensional structures (13). The ␣A-conotoxins, nevertheless, target neuromuscular receptors. A third subfamily of ␣-conotoxins known as the ␣4/7 subfamily has also been found; members of this subfamily target subtypes of neuronal and muscle nAChRs (14 -17). The ␣4/7 subfamily contains two disulfide bonds like the ␣3/5 subfamily but has a different spacing between the disulfide bonds.Even though a high resolutio...

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The .alpha.-Conotoxins GI and MI Distinguish between the Nicotinic Acetylcholine Receptor Agonist Sites while SI Does Not

Cited by 68 publications

References 34 publications

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

Molecular Interaction of α-Conotoxin RgIA with the Rat α9α10 Nicotinic Acetylcholine Receptor

Solution Conformation of α-Conotoxin EI, a Neuromuscular Toxin Specific for the α1/δ Subunit Interface of Torpedo Nicotinic Acetylcholine Receptor

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