TY - JOUR
T1 - Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit
AU - Akabas, Myles H.
AU - Kaufmann, Christine
AU - Archdeacon, Patrick
AU - Karlin, Arthur
N1 - Funding Information:
We thank Dr. Toni Claudio for the ACh receptor subunit cDNAs, Dr. Wayne Hendrickson for helpful discussions of protein structure, Drs. Barry Honig, Ranganathan Bharadwaj, and Anthony Nicholls for assistance in the use of the Molecular Modelling Facility for Molecular Biology at Columbia University (supported by NSF DlR-8720229), Dr. David Stauffer and Alex Fariborzian for the synthesis of the methanethiosulfonates, and Drs. Cynthia Czajkowski and Jonathan Javitch for comments on this paper. We also thank Gilda Salazar-Jimenez, Thomas Lin, and Ulla Beau-champ for technical assistance. This work was supported by research grants from the National Institutes of Health (NS07065), the American Heart Association, and the Muscular Dystrophy Association. M. H. A. is the recipient of a Klingenstein Award in the Neurosciences.
Copyright:
Copyright 2015 Elsevier B.V., All rights reserved.
PY - 1994/10
Y1 - 1994/10
N2 - Each residue in and flanking the M2 membrane-spanning segment of the α subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type β, γ, and δ 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 α 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.
AB - Each residue in and flanking the M2 membrane-spanning segment of the α subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type β, γ, and δ 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 α 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.
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U2 - 10.1016/0896-6273(94)90257-7
DO - 10.1016/0896-6273(94)90257-7
M3 - Article
C2 - 7524560
AN - SCOPUS:0027987062
VL - 13
SP - 919
EP - 927
JO - Neuron
JF - Neuron
SN - 0896-6273
IS - 4
ER -