Site Specificity of Agonist-Induced Opening and Desensitization of the Torpedo californica Nicotinic Acetylcholine Receptor
详细信息 查看全文
- 作者：Iraida E. Andreeva ; Selvanayagam Nirthanan ; Jonathan B. Cohen ; and Steen E. Pedersen
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：January 10, 2006
- 年：2006
- 卷：45
- 期：1
- 页码：195 - 204
- 全文大小：207K
- 年卷期：v.45,no.1(January 10, 2006)
- ISSN：1520-4995
文摘
Agonist-binding kinetics to the nicotinic acetylcholine receptor (AChR) from Torpedocalifornica were measured using sequential-mixing stopped-flow fluorescence methods to determine thecontribution 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 agonistconcentrations revealed four kinetic components: an initial, fast fluorescence decay, followed by a transientincrease, 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 KD values of2-4 
tities/mgr.gif">M for the desensitized AChR and ~600 tities/mgr.gif">M for the closed state. At this site, DC6C displayed astrongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer fromtryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned toDC6C dissociation from the 
ta.gif" BORDER=0 > site of the AChR in its closed conformation, on the basis of inhibitionwith the site-selective antagonists d-tubocurarine and -conotoxin MI. Fast decay amplitude data indicatedan apparent affinity of 0.9 tities/mgr.gif">M for the closed-state ta.gif" BORDER=0 > site; the closed-state 
-site affinity is inferred tobe near 100 tities/mgr.gif">M. These values and the known affinities for the desensitized conformation show that the site drives AChR desensitization to a ~40-fold greater extent than the ta.gif" BORDER=0 > site, undergoes energeticallylarger conformational changes, and is the primary determinant of agonist potency.
tities/mgr.gif">M for the desensitized AChR and ~600 tities/mgr.gif">M for the closed state. At this site, DC6C displayed astrongly blue-shifted emission spectrum, higher intrinsic fluorescence, and weaker energy transfer fromtryptophans than when bound to either agonist site. The initial, fast fluorescence decay was assigned toDC6C dissociation from the ta.gif" BORDER=0 > site of the AChR in its closed conformation, on the basis of inhibitionwith the site-selective antagonists d-tubocurarine and -conotoxin MI. Fast decay amplitude data indicatedan apparent affinity of 0.9 tities/mgr.gif">M for the closed-state ta.gif" BORDER=0 > site; the closed-state -site affinity is inferred tobe near 100 tities/mgr.gif">M. These values and the known affinities for the desensitized conformation show that the site drives AChR desensitization to a ~40-fold greater extent than the ta.gif" BORDER=0 > site, undergoes energeticallylarger conformational changes, and is the primary determinant of agonist potency.