Site specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor.
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 KD values of 2-4 microM for the desensitized AChR and approximately 600 microM 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 alphadelta site of the AChR in its closed conformation, on the basis of inhibition with the site-selective antagonists d-tubocurarine and alpha-conotoxin MI. Fast decay amplitude data indicated an apparent affinity of 0.9 microM for the closed-state alphadelta site; the closed-state alphagamma-site affinity is inferred to be near 100 microM. These values and the known affinities for the desensitized conformation show that the alphagamma site drives AChR desensitization to a approximately 40-fold greater extent than the alphadelta site, undergoes energetically larger conformational changes, and is the primary determinant of agonist potency.
Andreeva
Iraida E
IE
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77035, USA.
Nirthanan
Selvanayagam
S
Cohen
Jonathan B
JB
Pedersen
Steen E
SE
D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020916:Conotoxins / Q000494:pharmacology; D003619:Dansyl Compounds / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D018722:Nicotinic Agonists / Q000494:pharmacology*; D010622:Phencyclidine / Q000037:antagonists & inhibitors / Q000378:metabolism; D011335:Proadifen / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020916:Conotoxins; D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; C027933:(1-(5-dimethylaminonaphthalene)sulfonamido)-n-hexanoic acid-beta-N-trimethylammonium ethyl ester; C036155:conotoxin MI; D011335:Proadifen; D010622:Phencyclidine; D014403:Tubocurarine