The biochemistry of an acetylcholine receptor

Abstract

The acetylcholine receptor from Torpedo californica electroplax has been studied at three levels of molecular organization: receptor-rich membrane fragments, solubilized and purified receptor, and reconstituted receptor in phospholipid vesicles. The binding of cholinergic ligands t o the membrane-bound and the solubilized material is not cooperative, and the number of ligand sites is less than the number of toxin sites. In addition, the Durified macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified receptor and Torpedo phospholipids.

Specific binding of [3H]phencyclidines to membrane preparation

“…to [12], yielded linear curves ( fig.lB) Hence, we conclude that the phencyclidine derivatives under investigation bind at the PM level to specific binding sites present in the Torpedo membrane preparation which are not the cwBgt binding sites of the nicotinic receptors present in this preparation [4,6,15]. These data are in good agreement with the findings [l-3] that at FM levels phencyclidines do not block binding of 12'I_(yBgt.…”

“…We therefore decided to investigate the nature of this interaction by means of direct binding experiments using the 3H-labeled derivatives. The phencyclidines exhibit multiple interactions ( [l] and literature cited therein); their specific binding was studied in Torpedo electric organ since this enabled us to focus on the nicotinic cholinergic system (reviewed [4]). …”

Topographic studies of Torpedo acetylcholine receptor subunits as a transmembrane complex.

“…This indicates that, with respect to trypsin-sensitive residues, the 65,000-and 60,000-dalton subunits are the most exposed on the cytoplasmic face of the membrane, followed by the 50,000-dalton subunit, with the 40,000-dalton subunit exposed very little. More At the ultrastructural level, high-resolution electron micrographs of the AcChoR rosettes show from four to six similar "subunits" arranged about an electron-dense core (18,(48)(49)(50). This is consistent with recent reports that the receptor molecule consists of four homologous subunits present in the ratio 2:1:1:1 (31,32), for a total of five similar subunits, all exposed on the extracellular surface of the membrane.…”

“…Acad. Sci Examination of membrane vesicles by negative-stain electron microscopy showed no effect on the characteristic rosette structure of the AcChoR molecule (18,(48)(49)(50) either by the freeze-thawing process or by tryptic hydrolysis from either membrane face.…”

“…Except for one account (17), both species have been found to consist of four subunits of 40,000, 50,000, 60,000, and 65,000 daltons, with binding sites for a-neurotoxins, agonists, and some antagonists located on the 40,000-dalton subunit (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). These four subunits were found to possess a high incidence of amino acid sequence homology (31) and to exist in both membrane-bound and detergent-solubilized receptor as a pentameric complex with a stoichiometry of 2:1:1:1 (31,32).…”