Agonist-induced transitions of the acetylcholine receptor.
Anti-acetylcholine receptor (AChR) monoclonal antibody 383C binds to the beta-hairpin loop alpha(187-199) of only one of the two Torpedo AChR alpha subunits. The loop recognized is associated with the alpha subunit corresponding to the high-affinity d-tubocurarine (dTC) binding site. Desensitization of the receptor with carbamylcholine completely blocks the binding of 383C. Mild reduction of AChR alpha subunit cys 192-193 disulfide with DTT and subsequent reaction with 5-iodoacetamidofluorescein label only the high-affinity dTC alpha subunit. Rhodamine-labeled alpha-bungarotoxin (R-Btx) binds to the unlabeled AChR alpha subunit as monitored by fluorescence resonance energy transfer between the fluorescein and rhodamine dyes. A 10-A contraction of the distance between the dyes is observed following the addition of carbamylcholine. In a small angle X-ray diffraction experiment exploiting anomalous X-ray scattering from Tb(III) ions titrated into AChR Ca(II) binding sites, we find evidence for a change in the Tb(III) ion distribution in the region of the ion channel following addition of carbamylcholine to the AChR. The carbamylcholine-induced loss of the 383C epitope, the 10-A contraction of the beta-hairpin loop, and the loss of multivalent cations from the channel likely represent the first molecular transitions leading to AChR channel opening.
Fairclough
Robert H
RH
Department of Neurology, University of California, Davis, Davis, California 95616, USA. rhfairclough@ucdavis.edu
Agius
Mark A
MA
Gudipati
Eswari
E
Silvian
Laura
L
Hamaoka
Brent
B
Beltzner
Christopher C
CC
Lin
Mike Y
MY
Chuang
Anthony R
AR
Richman
David P
DP
D000818:Animals; D001665:Binding Sites; D002217:Carbachol / Q000737:chemistry; D018679:Cholinergic Agonists / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D066298:In Vitro Techniques; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000276:immunology; D014961:X-Ray Diffraction / Q000295:instrumentation / Q000379:methods
D018679:Cholinergic Agonists; D021122:Protein Subunits; D011950:Receptors, Cholinergic; D002217:Carbachol