Document Detail


[3H]Benzophenone photolabeling identifies state-dependent changes in nicotinic acetylcholine receptor structure.
MedLine Citation:
PMID:  17685589     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Interactions of benzophenone (BP) with the Torpedo nicotinic acetylcholine receptor (nAChR) were characterized by electrophysiological analyses, radioligand binding assays, and photolabeling of nAChR-rich membranes with [3H]BP to identify the amino acids contributing to its binding sites. BP acted as a low potency noncompetitive antagonist, reversibly inhibiting the ACh responses of nAChRs expressed in Xenopus oocytes (IC50 = 600 microM) and the binding of the noncompetitive antagonist [3H]tetracaine to nAChR-rich membranes (IC50 = 150 microM). UV irradiation at 365 nm resulted in covalent incorporation of [3H]BP into the nAChR subunits (delta > alpha approximately beta > gamma), with photoincorporation limited to the nAChR transmembrane domain. Comparison of nAChR photolabeling in the closed state (absence of agonist) and desensitized state (equilibrated with agonist) revealed selective desensitized state labeling in the delta subunit of deltaPhe-232 in deltaM1 and deltaPro-286/deltaIle-288 near the beginning of deltaM3 that are within a pocket at the interface between the transmembrane and extracellular domains. There was labeling in the closed state within the ion channel at position M2-13 (alphaVal-255, betaVal-261, and deltaVal-269) that was reduced by 90% upon desensitization and labeling in the transmembrane M3 helices of the beta and gamma subunits (betaMet-285, betaMet-288, and gammaMet-291) that was reduced by 50-80% in the desensitized state. Labeling at the lipid interface (alphaMet-415 in alphaM4) was unaffected by agonist. These results provide a further definition of the regions in the nAChR transmembrane domain that differ in structure between the closed and desensitized states.
Authors:
Galo Garcia; David C Chiara; Selvanayagam Nirthanan; Ayman K Hamouda; Deirdre S Stewart; Jonathan B Cohen
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2007-08-09
Journal Detail:
Title:  Biochemistry     Volume:  46     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  2007 Sep 
Date Detail:
Created Date:  2007-09-04     Completed Date:  2007-10-10     Revised Date:  2007-12-03    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  10296-307     Citation Subset:  IM    
Affiliation:
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Benzophenones / metabolism*,  pharmacology
Binding Sites
Cell Membrane / drug effects,  metabolism
Ion Channels / metabolism
Nicotinic Agonists / pharmacology
Nicotinic Antagonists / pharmacology
Peptide Fragments / metabolism
Photoaffinity Labels / analysis*,  metabolism
Protein Structure, Secondary
Protein Structure, Tertiary
Protein Subunits / chemistry,  metabolism
Receptors, Nicotinic / chemistry*,  metabolism*
Torpedo / metabolism*
Tritium / metabolism*
Xenopus
Grant Support
ID/Acronym/Agency:
GM-58448/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Benzophenones; 0/Ion Channels; 0/Nicotinic Agonists; 0/Nicotinic Antagonists; 0/Peptide Fragments; 0/Photoaffinity Labels; 0/Protein Subunits; 0/Receptors, Nicotinic; 10028-17-8/Tritium; 119-61-9/benzophenone

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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