The Acetylcholine Receptor as Part of a Protein Complex in Receptor-Enriched Membrane Fragments from Torpedo californica Electric Tissue

Abstract

The acetylcholine receptor from Torpedo californicu electric tissue consisting of polypeptide chains of molecular weight 42 000 (k 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270 000 ( 2 30 000). Besides the receptor subunit (a-chain), at least three further classes of polypeptide chains are part of the complex : ( M , 48 000), y ( M , 62 000) and 6 ( M , 68 000).This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent : From the 270 000 molecular weight particle the four predominant polypeptide chains of the membrane, a, p, y, and 6, can be obtained.The &polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.The nicotinic acetylcholine receptor protein is believed to be involved in the ion permeability changes of the postsynaptic membrane of cholinergic synapses induced by the transmitter acetylcholine. Elucidation of the mechanism of the events in the membrane during opening and closing of ion gates is expected from the analysis of the structure and function of the molecules comprising the membrane. Relatively few protein species appear to be present. Their intensive investigation in recent years has provided a large amount of information (for a collection of recent reviews see [l]), but most of the basic questions remain unanswered. It is still unknown if the receptor, i.e. the transmitterbinding protein, is simultaneously the ion pore and if its conformation determines the ion permeability of the membrane. Most laboratories tackled this question by means of reconstitution experiments with the purified binding protein [2-51 but the results are equivocal. Another approach is to investigate the function of the various components by means of affinity labeling [6,7]. This approach has shown that not all components contain binding sites for cholinergic ligands. A polypeptide chain of molecular weight 40 000 contains the binding sites for compounds with quaternary ammonium groups [6] and for a-neurotoxin from cobra venom [7]. This polypeptide chain appears to represent A preliminary communication has been prescnted at a meeting of the Gesell,sc.hqft ,fur Biologische Chemir [34]. the acetylcholine receptor, but its relationship to the other chains detectable in the membrane remains unclear.The acetylcholine receptor from the electric organ of Electroplzorus electricus or Torpedo culifornicu appears to be part of an oligomeric protein complex of several different polypeptide chains [7 -141. After extraction with non-ionic detergents and purification by affinity chromatography, the protein from Electrophorus has been shown to be possibly a pentamer of two different polypeptide chains [9]. Other authors obtained preparations with three different chains [16]. For the protein from Torpedo, one [ 15, 171, two [ 1 1 , 181, three [ 191, and four [ l6,20,7] different polypeptide chains hav...

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

“…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). This stoichiometry dictates a size of 255,000 daltons for the AcChoR monomer, in agreement with the experimentally determined values of 250,000-270,000 daltons (33)(34)(35). In light of this molecular definition of the AcChoR and of its defined function alluded to above, it is of interest to determine the topography of its subunits in the membrane and eventually to assign a specific function to each polypeptide.…”

The nicotinic acetylcholine receptor and its ion channel

“…All the polypeptide chains are glycosylated (see below) and the true M , of the protein part, as calculated from the amino acid sequences determined by cloning and sequencing cDNAs encoding the respective subunit precursors [9 -1 I], are 50200 (a), 53 700 (p), 56 300 ( y ) and 57 600 (6), yielding an M , for the nAChR protein of 268000. Adding to this an estimated M , 20000 for the carbohydrate moiety, the total M , is about 290000, a value close to that obtained previously from laser scattering data (292 000 [ 121) or SDS/polyacrylamide gel electrophoresis of cross-linked nAChR (270000 [7]). …”

“…The nAChR from electroplaque membranes from electric fish as purified by affinity chromatography [3-51 is a pentameric protein [6,7] composed of five polypeptide chains of four types. The resulting quaternary structure is a2, fl?…”

Gαq-Coupled Muscarinic Acetylcholine Receptors Enhance Nicotinic Acetylcholine Receptor Signaling inCaenorhabditis elegansMating Behavior

“…nAChRs are ligand-gated cation channels that consist of five subunits and were first identified by their sensitivity to the agonist nicotine (Langley, 1907;Chang et al, 1977;Hucho et al, 1978;ContiTronconi and Raftery, 1982). mAChRs are seventransmembrane G-protein-coupled receptors, which were identified by their sensitivity to muscarine (Dale, 1914;Ewins, 1914;Kubo et al, 1986;Lanzafame et al, 2003).…”