ANNULAR AND NONANNULAR BINDING-SITES FOR CHOLESTEROL ASSOCIATED WITH THE NICOTINIC ACETYLCHOLINE-RECEPTOR
ANNULAR AND NONANNULAR BINDING-SITES FOR CHOLESTEROL ASSOCIATED WITH THE NICOTINIC ACETYLCHOLINE-RECEPTOR
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DOI：
10.1021/bi00407a018
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发表时间：
1988-04-05
期刊：
影响因子：
2.9
通讯作者：
MCNAMEE, MG
中科院分区：
文献类型：
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作者：
JONES, OT;MCNAMEE, MG
脂质和烟碱乙酰胆碱受体之间的相互作用的鱼雷加州已被测定在重组膜含有纯化的受体和定义的脂质。溴化脂质部分淬灭乙酰胆碱受体的固有荧光的能力已被用于监测蛋白质与周围脂质之间的接触。通过使用伦敦和Feigenson [伦敦，E.，和Feigenson，G. W.（1981）Biochemistry 20，1939-1048]和Simmonds等人[Simmonds，A. C.的方法，鲁尼，E。K.，和Lee，A. G.（1984）Biochemistry 23，1432-1441]。二油酰磷脂酰胆碱及其二溴衍生物是用于重建膜以建立淬灭参数的两种主要脂质。胆固醇和磷脂酰胆碱之间的竞争研究表明，胆固醇不能有效地竞争被推测为包围受体（环状脂质）的膜包埋部分的磷脂位点。然而，二溴胆固醇部分淬灭受体，并导致在纯二溴磷脂酰胆碱膜的受体的额外淬灭。结果是一致的存在下，额外的结合位点的胆固醇是不接近磷脂（nonannular网站）。通过使用胆固醇半琥珀酸酯及其二溴类似物获得了类似的结果，这两者都可以比胆固醇更容易地引入膜中，因为它们在水中的溶解度更大。脂肪酸似乎竞争环状和非环状位点，淬灭数据的分析表明，有5-10个非环状位点与受体相关。胆固醇已被证明在乙酰胆碱受体结构稳定和离子通道活性中起着关键作用，这里提供的结果提供了有关胆固醇-受体相互作用的额外信息。
Interactions between lipids and the nicotinic acetylcholine receptor for Torpedo californica have been measured in reconstituted membranes containing purified receptor and defined lipids. The ability of brominated lipids to partially quench the intrinsic fluorescence of the acetylcholine receptor has been exploited to monitor contacts between the protein and the surrounding lipid. Relative bindings constants for lipid binding to the protein have been quantitatively determined by measuring quenching observed in mixtures of brominated and nonbrominated lipids by use of equilibrium excharge equations developed by London and Feigenson [London, E., and Feigenson, G. W. (1981) Biochemistry 20, 1939-1048] and by Simmonds et al. [Simmonds, A. C., Rooney, E. K., and Lee, A. G. (1984) Biochemistry 23, 1432-1441]. Dioleoylphosphatidylcholine and its dibromo derivative are the two prinicpal lipids used in the reconstituted membranes to establish the quenching parameters. Competition studies between cholesterol and phosphatidylcholine indicate that cholesterol does not compete effectively for the phospholipid sites presumed to surround the membrane-embedded portions of the receptors (annular lipids). However, dibromocholesterol partially quenches the receptor and leads to additional quenching of receptor in pure dibromophosphatidylcholine membranes. The results are consistent with the presence of additional binding sites for cholesterol that are not accessible to phsopholipids (nonannular sites). Similar results are obtained by using cholesterol hemisuccinate and its dibromo analogue, both of which can be introduced into membranes more easily than cholesterol because of their greater solubility in water. Fatty acids appear to compete for both annular and nonannular sites, and analysis of the quenching data suggests that there are 5-10 nonannular sites associated with the receptor. Cholesterol has been shown to play a critical role in both acetylcholine receptor structural stabilization and ion channel activity, and the results presented here provide additional information about cholesterol-receptor interactions.