Location of functional regions of acetylcholine receptor α-subunit by site-directed mutagenesis

Abstract

The availability of cloned cDNAs encoding the four subunits of the Torpedo acetylcholine receptor, which can be expressed to make functional receptors in Xenopus oocytes, has made possible a detailed investigation of the functions of the different structural components of the receptor. The functional analysis of receptors with alpha-subunits altered at specific sites by site-directed mutagenesis of the cDNA has allowed the location of specific regions of the alpha-subunit molecule involved in acetylcholine binding and forming a transmembrane ionic channel.

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

“…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.…”

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity

“…However, a fundamental question remains concerning the functional specificity of individual ACh receptor subunits in channel function. The 01 subunits contain the two ACh binding sites (Kao et al, 1984;Mishina et al, 1985) and it is thought that LV@~ and 6 subunits all contribute to the structure of the cation channel (Changeux et al, 1984).…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…This was achieved by a somewhat indirect route: cultured mammalian (COS) cells were first transfected with nAChR subunit cDNA constructs, after which the transcribed cRNAs were then isolated from COS cells and microinjected into Xenopus oocytes. In later studies, functional expression was achieved in Xenopus oocytes using cRNA that had been transcribed in vitro from nAChR subunit cDNAs, using purified RNA polymerase [80]. In addition, a more direct approach of injecting cDNA directly into the oocyte nucleus has also been used successfully for the functional expression of nAChRs, first being used to characterize the neuronal α4β2 nAChR [81].…”

“…In 1985, the use of site-directed mutagenesis combined with heterologous expression enabled the identification of regions and individual amino acids within the Torpedo α subunit that influence ligand binding and functional expression [80]. This was the first of many hundreds of studies that have employed site-directed mutagenesis to characterize nAChRs.…”

“…Site-directed mutagenesis, in combination with heterologous expression, has however been used successfully to examine phenomena such as, subunit glycosylation [225][226][227], the role of disulfide-linked cysteines [80,228], cell surface receptor trafficking [214,229,230], interactions with agonists and antagonists [231][232][233], modulation by zinc [234,235] and by other allosteric modulators [199,200], calcium permeability [236] and channel gating [202,[237][238][239][240][241]. Analysis of mutated nAChRs has also provided insights into how subunit domains may move during receptor activation [241,242].…”