Low chemical specificity of the nicotinic acetylcholine receptor sterol activation site

被引:35
作者
Addona, GH
Sandermann, H
Kloczewiak, MA
Miller, KW
机构
[1] Massachusetts Gen Hosp, Dept Anesthesia & Crit Care, Boston, MA 02114 USA
[2] Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
[3] GSF Forschungszentrum Umwelt & Gesundheit GMBH, Inst Biochem Pflanzenpathol, D-85764 Neuherberg, Germany
来源
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES | 2003年 / 1609卷 / 02期
关键词
acetylcholine receptor; reconstitution; gating; cholesterol; steroid specificity;
D O I
10.1016/S0005-2736(02)00685-5
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:177 / 182
页数:6
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