Anti-acetylcholine receptor (AChR) monoclonal antibody 383C binds to the β-hairpin loop α(187-199) of only one of the two Torpedo AChR α subunits. The loop recognized is associated with the α 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 α subunit cys 192-193 disulfide with DTT and subsequent reaction with 5-iodoacetamidofluorescein label only the high-affinity dTC α subunit. Rhodamine-labeled α-bungarotoxin (R-Btx) binds to the unlabeled AChR α subunit as monitored by fluorescence resonance energy transfer between the fluorescein and rhodamine dyes. A 10-Å 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-Å contraction of the β-hairpin loop, and the loss of multivalent cations from the channel likely represent the first molecular transitions leading to AChR channel opening.
- Acetylcholine receptor (AChR)
- D-tubocurarine (dTC)
- X-ray diffraction
- X-ray scattering
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)