Details

Autor(en) / Beteiligte

• Andreeva, Iraida E
• Nirthanan, Selvanayagam
• Cohen, Jonathan B
• Pedersen, Steen E

Titel

Site Specificity of Agonist-Induced Opening and Desensitization of the Torpedo californica Nicotinic Acetylcholine Receptor

Ist Teil von

• Biochemistry (Easton), 2006-01, Vol.45 (1), p.195-204

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

2006

Quelle

MEDLINE

Beschreibungen/Notizen

• Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were measured using sequential-mixing stopped-flow fluorescence methods to determine the contribution of each individual site to agonist-induced opening and desensitization. Timed dansyl-C6-choline (DC6C) binding followed by its dissociation upon mixing with high, competing agonist concentrations revealed four kinetic components:  an initial, fast fluorescence decay, followed by a transient increase, and then two characteristic decays that reflect dissociation from the desensitized agonist sites. The transient increase resulted from DC6C binding to the open-channel based on its prevention by proadifen, a noncompetitive antagonist. Further characterization of DC6C channel binding by the inhibition of [3H]phencyclidine binding and by equilibrium measurements of DC6C fluorescence yielded K D values of 2−4 μM for the desensitized AChR and ∼600 μM for the closed state. At this site, DC6C displayed a strongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer from tryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned to DC6C dissociation from the αδ site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and α-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 μM for the closed-state αδ site; the closed-state αγ-site affinity is inferred to be near 100 μM. These values and the known affinities for the desensitized conformation show that the αγ site drives AChR desensitization to a ∼40-fold greater extent than the αδ site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.