Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist

• Published in: The Journal of biological chemistry Vol. 267; no. 22; pp. 15770 - 15783
• Main Authors: WHITE, B. H, COHEN, J. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.08.1992
• Subjects: acetylcholine; agonists; Amino Acid Sequence; Animals; Azirines - metabolism; binding; Binding Sites; Biological and medical sciences; Carbachol - pharmacology; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Cyanogen Bromide; Electric Organ - metabolism; Fundamental and applied biological sciences. Psychology; Iodine Radioisotopes; Macromolecular Substances; Models, Structural; Molecular and cellular biology; Molecular Sequence Data; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Peptide Fragments - isolation & purification; Protein Conformation; receptors; Receptors, Nicotinic - drug effects; Receptors, Nicotinic - metabolism; structure; Torpedo; Trypsin; Membrane receptor; Vertebrata; Agonist; Cholinergic receptor; Molecular structure; Pisces; Torpedo californica; Photoaffinity labelling; Subunit; Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)49602-6

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.