title: Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site.
abstract: The nicotinic acetylcholine receptor (nAcChoR) has an absolute requirement for cholesterol if agonist-stimulated channel opening is to occur [Biochemistry 25 (1986) 830]. Certain non-polar analogs could replace cholesterol in vectorial vesicle permeability assays. Using a stopped-flow fluorescence assay to avoid the limitations of permeability assays imposed by vesicle morphology, it was shown that polar conjugates of cholesterol could also satisfy the sterol requirement [Biochim. Biophys. Acta 1370 (1998) 299]. Here this assay is used to explore the chemical specificity of sterols. Affinity-purified nAcChoRs from Torpedo were reconstituted into bilayers at mole ratios of 58:12:30 [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)/steroid]. When the enantiomer of cholesterol was used, or when the stereochemistry at the 3-hydroxy group was changed from beta to alpha by substituting epicholesterol for cholesterol, activation was still supported. The importance of cholesterol's planar ring structure was tested by comparing planar cholestanol (5alpha-cholestan-3beta-ol) with nonplanar coprostanol (5beta-cholestan-3beta-ol). Both supported activation. Thus, these steroids support activation independent of structural features known to be important for modulation of lipid bilayer properties. This provides indirect support for a steroid binding site possessing very lax structural requirements.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: George H
authors.initials: GH
authors.lastname: Addona
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sandermann
authors.forename: Marek A
authors.initials: MA
authors.lastname: Kloczewiak
authors.forename: Keith W
authors.initials: KW
authors.lastname: Miller
mesh_terms: D000818:Animals; D004083:Cholestanol / Q000031:analogs & derivatives* / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D013261:Sterols / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014101:Torpedo
chemical_list: D010713:Phosphatidylcholines; D011978:Receptors, Nicotinic; D013261:Sterols; D004083:Cholestanol; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine