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dc.contributor.authorAndreeva, IE
dc.contributor.authorNirthanan, S
dc.contributor.authorCohen, JB
dc.contributor.authorPedersen, SE
dc.date.accessioned2017-05-03T15:29:00Z
dc.date.available2017-05-03T15:29:00Z
dc.date.issued2006
dc.date.modified2013-12-12T03:39:27Z
dc.identifier.issn0006-2960
dc.identifier.doi10.1021/bi0516024
dc.identifier.urihttp://hdl.handle.net/10072/54920
dc.description.abstractAgonist-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.
dc.description.peerreviewedYes
dc.description.publicationstatusYes
dc.languageEnglish
dc.language.isoeng
dc.publisherAmerican Chemical Society
dc.publisher.placeWashington, DC
dc.relation.ispartofstudentpublicationN
dc.relation.ispartofpagefrom195
dc.relation.ispartofpageto204
dc.relation.ispartofissue1
dc.relation.ispartofjournalBiochemistry
dc.relation.ispartofvolume45
dc.rights.retentionY
dc.subject.fieldofresearchMedicinal and biomolecular chemistry
dc.subject.fieldofresearchBiochemistry and cell biology
dc.subject.fieldofresearchMedical biochemistry and metabolomics
dc.subject.fieldofresearchBasic pharmacology
dc.subject.fieldofresearchcode3404
dc.subject.fieldofresearchcode3101
dc.subject.fieldofresearchcode3205
dc.subject.fieldofresearchcode321401
dc.titleSite-specificity of agonist-induced opening and desensitization of the Torpedo californica nicotinic acetylcholine receptor.
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.rights.copyrightSelf-archiving of the author-manuscript version is not yet supported by this journal. Please refer to the journal link for access to the definitive, published version or contact the author[s] for more information.
gro.date.issued2006
gro.hasfulltextNo Full Text
gro.griffith.authorNirthanan, Niru


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