title: Site of resting state inhibition of the nicotinic acetylcholine receptor by a hydrophobic inhibitor.
abstract: The lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) is a noncompetitive, resting-state inhibitor of the nicotinic acetylcholine receptor (nAChR) that requires tens of milliseconds of preincubation to inhibit agonist-induced cation efflux. At equilibrium, [(125)I]TID photoincorporates into both the ion channel and the lipid-protein interface of the Torpedo nAChR. To determine which of these regions is responsible for resting-state inhibition, we characterized the interactions between [(125)I]TID and nAChR-rich membranes milliseconds after mixing, by use of time-resolved photolabeling. Photolabeling was performed after preincubation times of 2 ms or 600 s (equilibrium), and the efficiencies of incorporation at specific residues were determined by amino-terminal sequence analysis of nAChR-subunit proteolytic fragments isolated by SDS-PAGE and/or reversed-phase HPLC. Equilibration of TID with lipid was complete within a millisecond as determined by both stopped-flow fluorescence quenching of diphenylhexatriene in lipid bilayers and photoincorporation into nAChR-rich membrane phospholipids. Equilibration with the lipid-protein interface (alphaM4) was slightly slower, reaching approximately 50% that at equilibrium after 2 ms preincubation. In contrast, equilibration with the channel region (alpha 2 and deltaM2) was much slower, reaching only 10% that at equilibrium after 2 ms preincubation. Within the ion channel, the ratio of [(125)I]TID incorporation between M2 residues 9', 13', and 16' was independent of preincubation time. We conclude that TID's access to the ion channel is more restricted than to the lipid-protein interface and that TID bound within the ion channel is responsible for flux inhibition upon activation of the nAChR.
authors.affiliation: Department of Anesthesia and Critical Care, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114, USA.
authors.forename: D C
authors.initials: DC
authors.lastname: Chiara
authors.forename: M A
authors.initials: MA
authors.lastname: Kloczewiak
authors.forename: G H
authors.initials: GH
authors.lastname: Addona
authors.forename: J A
authors.initials: JA
authors.lastname: Yu
authors.forename: J B
authors.initials: JB
authors.lastname: Cohen
authors.forename: K W
authors.initials: KW
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001389:Azirines / Q000378:metabolism / Q000494:pharmacology*; D004161:Diphenylhexatriene / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D018733:Nicotinic Antagonists / Q000378:metabolism* / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014101:Torpedo
chemical_list: D001389:Azirines; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D008051:Lipid Bilayers; D018733:Nicotinic Antagonists; D010446:Peptide Fragments; D010743:Phospholipids; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D004161:Diphenylhexatriene; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine