The reaction site of a non-competitive antagonist in the delta-subunit of the nicotinic acetylcholine receptor.

Abstract

A site in the primary structure of the nicotinic acetylcholine receptor from Torpedo marmorata covalently labeled with the non-competitive antagonist [3H]triphenylmethylphosphonium (TPMP+) was localized. The label was found in position 262 of the 6-polypeptide chain. This site is specifically labeled in the presence of the agonist carbamoylcholine. Labeling is prevented by the non-competitive antagonist histrionicotoxin. Position 262, probably a serine, is located in the highly conserved membrane-spanning helix M2 (according to the predicted folding scheme of Finer-Moore and Stroud (1984). The relationship of this site to the receptor's ion channel and its regulation is discussed.

The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

“…The results of covalent labeling with CPZ and triphenylmethylphosphonium provide convergent evidence that segments M2 are potential components of the ion translocation device. Consistent with this view, site-directed mutagenesis experiments have shown, following the original observation of Giraudat et al [I] and Oberthiir et al [13], that a region of the ~-subunit comprising segment M2 and the portion between segments M2 and M3 contribute to the regulation of ion transport [32]. More recent works from the same groups have shown that clusters of charged residues neighbouring both ends of segment M2 of the a-, fl-, 7"-and d-subunits are major determinants of the rate of ion transport [33].…”

“…Furthermore, electrophysiological (review in [27][28][29]) as well as biochemical (review in [6,30]) evidence supports the view that the high-affinity NCB site is located within the ion channel. Photoactivatable NCBs are thus useful tools for direct identification of potential channel-forming elements within each of the AChR subunits [1,2,12,13].…”

“…Under equilibrium conditions, NCBs reversibly interact with a few categories of belong to the hydrophobic segment M2 of the fland 8-chains (residues ~?-Ser-254 and fl-Leu-257, 8-Ser-262) [1,2]. Also, 8-Ser-262 [13] and homologous residues of the or-and g-chains [12] are labeled by the NCB [3H]triphenylmethylphosphonium. These data thus support the proposal that the unique high-affinity NCB site is delimited by homologous regions of the five subunits, which include segment M2, where all the labeled residues identified so far are located.…”

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits

“…The procedure was essentially as described before [6]. The photolabeled receptor-rich membranes were dissolved in sample buffer and the polypeptide chains were separated in two steps by preparative SDS-polyacrylamide gel electrophoresis, using the apparatus from BRL, Bethesda.…”

“…Noncompetitive antagonists, a very heterogenous group of compounds, are thought to be tools for its elucidation because they block ion fluxes by sterically or allosterically interacting with the channel [4]. Recently a binding site for the noncompetitive antagonists chlorpromazine [5] and TPMP+ [6] has been localized in the S-subunit of AChR from Torpedo marmorata by photoaffinity labeling and microsequencing work. It was identified as serine 262 located in the membranespanning helix II (according to the five-helix model proposed by Finer-Moore and Stroud [7] and Guy [S]).…”