Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor

Abstract

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.

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

“…Do~-response curves for agonist (Fig. 3) indicated that mutation of %'187 into F resulted in a 10-fold decrease in ACh apparent affinity (Table i), a result which is in good agreement with the recently described mutation of the homologous residue from mouse muscle nicotinic receptor a-subunit [8].…”

“…As observed for mutations near amino acid Y187 in mouse muscle receptor [8], several mutations near Y92 and W!48 of the ~7-subunit, like $94N and WI53F (Fig. 2), G82E and GI51D (not shown), did not cause significant changes on the physiological properties of the receptor.…”

“…These sites are mainly carried by the ~-subunits and include a doublet ofcysteine (C) residues (CI92, CI93 in Torpedo receptor) which react with sulfhydryl reagents after reduction of the receptor [5], Further studies based on cz-bungarotoxin binding to a-subunit peptide fragments (reviewed in [4]), and the consequences on re~ptor channel response of disulfide bridges reduction (reviewed in [6]) and point mutations [7,8], established that the region enlgO-200, containing Ylg0, C!92 and CI93, contribute to the recognition of cholinergie ligands.…”

Nicotinic Acetylcholine Receptors

“…This tyrosine is on the C-loop and is close to the pair of adjacent cysteine residues (C192 and C193) that characterises α subunits. The mutation Y190F (like Y93F and W149F) also decreases macroscopic ACh binding (Tomaselli et al, 1991), and increases the EC 50 by 184-fold (in embryonic mouse muscle receptor, Chen et al, 1995). These effects were originally attributed to changes in the binding.…”

The functional architecture of the acetylcholine nicotinic receptor explored by affinity labelling and site-directed mutagenesis

“…As mentioned, mutations of the MBTA and DDF-labelled Cysi92 and 193 into serines, interfere with a-bungarotoxin binding and with the response to acetylcholine . Also, in the same domain mutation of Tyri9O into Phe yields a receptor which requires more than 50-fold higher concentration of acetylcholine for channel activation than wild type channels with the correlative decrease of agonist-binding affinity (Tomaselli et al 1991).…”