Agonist‐Induced Transitions of the Acetylcholine Receptor

Abstract

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.

Ligand-Induced Conformational Change in the α7 Nicotinic Receptor Ligand Binding Domain

“…This ligand-bound structure should correspond to either an active or desensitized state for nAChRs. Upon agonist binding, the residues of the binding site are believed to form an aromatic cage (Lester et al, 2004), drawing the C-loop toward the binding site (Karlin, 2002;Fairclough et al, 2003), a motion particularly evident by comparing the recent AChBP crystal structures with and without carbamoylcholine (Celie et al, 2004). This C-loop motion in turn somehow propagates downward toward the TMD (Grosman et al, 2000;Chakrapani et al, 2004).…”