Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site‐directed mutagenesis

Galzi, Jean‐Luc; Bertrand, Daniel; Devillers‐Thiéry, Anne; Revah, Frédéric; Bertrand, Sonia; Changeux, Jean‐Pierre

doi:10.1016/0014-5793(91)80668-s

Cited by 159 publications

(98 citation statements)

References 23 publications

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“…Curiously, there was no impact of mutation of Trp-149. However, this residue has been found to play a primary role in agonist binding to chicken ␣7-nAChRs (44). As noted previously, this tryptophan lies on the other side of the agonist binding pocket from Tyr-188, on the basis of the AChBP structure, and thus may not make contact with A␤.…”

Section: Discussionmentioning

confidence: 83%

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Tong

Arora

White

et al. 2011

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 83%

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Tong

Arora

White

et al. 2011

Journal of Biological Chemistry

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“…The binding site for orthosteric agonists, such as acetylcholine, is well established and mutations located at this site in α7 nAChRs have been shown to cause a substantial rightward shift in the dose-response curve for acetylcholine (25). Here, we examined the influence of one such mutation (W148F), located close to the known binding site of acetylcholine.…”

Section: Resultsmentioning

confidence: 99%

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Gill

Savolainen

Young

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

118

194

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Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular "orthosteric" binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site.allosteric potentiation | neurotransmitter receptor

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“…Upon binding of ligands lacking the capacity for FRET, tryptophan fluorescence quenching has also been observed for acetylcholinesterase (9); its binding site is at the base of a narrow gorge whose base and walls are lined with aromatic residues (10). In the muscle and ␣7 nAChR, residue substitution of tryptophans homologous to residues 53 and 143 reduces ligand affinity, but does not eliminate binding (11,12), and a charge-transfer complex involving one or more tryptophans has been proposed to stabilize various ligand complexes (13,14). Systematic substitution of other aromatic residues for the five tryptophans should enable one to delineate further their individual contributions to quenching of fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Tryptophan Fluorescence Reveals Conformational Changes in the Acetylcholine Binding Protein

Hansen¹,

Radić²,

Talley³

et al. 2002

Journal of Biological Chemistry

123

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The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the ␣-neurotoxins. Since agonist binding occurs in millisecond time frames, and the ␣-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.Ligand-gated ion channels, of which the nicotinic acetylcholine receptor is a prototypic structure, are composed of five subunits whose ␣-carbon chains traverse the membrane four times (1, 2); their hydrophobicity and size preclude conventional structural studies at atomic resolution by x-ray crystallography or nuclear magnetic resonance spectrometry. Recently, an acetylcholine binding protein (AChBP) 1 from the fresh water snail, Lymnaea stagnalis, has been characterized, crystallized, and its structure determined (3, 4). The crystal structure shows virtually all of the features predicted from a host of affinity labeling, site-specific mutagenesis, and subunit assembly studies conducted on the nicotinic receptor for over 2 decades (1, 2, 5). Although the isolated protein shares ligand recognition characteristics with its closest mammalian homolog, the pentameric ␣7 receptor (4), details on its ligand specificity, binding kinetics, and conformational changes remain unknown. These questions are critical to ascertaining whether the snail protein has the recognition properties and conformational states to serve as a functional as well as a structural surrogate of the extracellular domain of the nicotinic receptor. To this end, we have expressed the binding protein in a mammalian system from a chemically synthesized cDNA of 637 bp. The cDNA contains restriction sites at various locations to allow for substitution of encoding receptor segments into the cDNA template of the binding protein. Upon ligand binding, AChBP shows major changes in fluorescence emitted from five tryptophans on each subunit, providing an i...

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Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site‐directed mutagenesis

Cited by 159 publications

References 23 publications

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Tryptophan Fluorescence Reveals Conformational Changes in the Acetylcholine Binding Protein

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