title: Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.
abstract: Each residue in and flanking the M2 membrane-spanning segment of the alpha subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Cysteines substituted for Glu-262, Leu-258, Val-255, Ser-252, Leu-251, Leu-250, Ser-248, Leu-245, Thr-244, and Glu-241 reacted with the positively charged, hydrophilic, sulfhydryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), added extracellularly. These 10 residues, therefore, are exposed in the channel lumen. The pattern of exposure is compatible with an alpha helix, interrupted by an extended structure from Leu-250 to Ser-252. Acetylcholine caused subtle changes in the accessibilities of some of the engineered cysteines. Since all 10 residues are accessible to MTSEA in the closed state of the channel, the channel gate is at least as cytoplasmic as Glu-241, the most cytoplasmic of the residues tested.
authors.affiliation: Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
authors.forename: M H
authors.initials: MH
authors.lastname: Akabas
authors.forename: C
authors.initials: C
authors.lastname: Kaufmann
authors.forename: P
authors.initials: P
authors.lastname: Archdeacon
authors.forename: A
authors.initials: A
authors.lastname: Karlin
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry*; D008698:Mesylates / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics; D011978:Receptors, Nicotinic / Q000737:chemistry; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents
chemical_list: D007473:Ion Channels; D008698:Mesylates; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D013439:Sulfhydryl Reagents; C074502:methanethiosulfonate; D003545:Cysteine; D000109:Acetylcholine