Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol
Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol
复制标题
DOI：
10.1016/s0005-2736(98)00153-9
复制
发表时间：
1998-11-11
影响因子：
3.4
通讯作者：
Blanton, MP
中科院分区：
文献类型：
--
作者：
Corbin, J;Wang, HH;Blanton, MP
胆固醇的新型光反应类似物3 α-（4-叠氮基-3-[I-125]碘水杨酸）-胆甾-5-3 n3（[I-125]叠氮基-胆固醇）被用于标记来自加利福尼亚电鳐的天然乙酰胆碱受体（AChR）丰富的膜和亲和纯化的重组成脂质囊泡的电鳐AChR。在这两种情况下，所有四个乙酰胆碱受体亚单位纳入[I-125]叠氮基胆固醇等摩尔的基础上，既不模式，也不标记的程度受到影响的激动剂氨甲酰胆碱的存在下。在每个AChR亚基的标记区域最初映射由金黄色葡萄球菌V8蛋白酶消化的大片段，其中包含AChR跨膜段。通过对V8蛋白酶亚基片段α V8-20（α Ser-173-Glu-338）、α V8-10（α Asn-339-Gly-439）和γ V8-14（γ Leu-373-Pro-489）进行彻底胰蛋白酶消化，进一步绘制[I-125]叠氮基胆固醇掺入位点。通过反相高效液相色谱法分离这些肽，并通过氨基末端序列分析鉴定标记肽。[I-125]叠氮基胆固醇标记定位于几乎仅包含α-M4、α-M1和γ-M4跨膜片段的肽。这些结果表明胆固醇的结合域位于AChR的脂质-蛋白质界面。(C)1998 Elsevier Science B. V.保留所有权利。
A novel photoreactive analog of cholesterol, 3 alpha-(4-azido-3-[I-125]iodosalicylic)-cholest-5-3n3 ([I-125]azido-cholesterol), was used to label both native acetylcholine receptor (AChR)-rich membranes from Torpedo californica and affinity-purified Torpedo AChRs reconstituted into lipid vesicles. In both cases all four AChR subunits incorporated [I-125]azido-cholesterol on an equal molar basis and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine. Labeled regions in each of the AChR subunits were initially mapped by Staphylococcus aureus V8 protease digestion to large fragments which contain the AChR transmembrane segments. Sites of [I-125]azido-cholesterol incorporation were further mapped by exhaustive tryptic digestion of the V8 protease subunit fragments alpha V8-20 (alpha Ser-173-Glu-338), alpha V8-10 (alpha Asn-339-Gly-439), and gamma V8-14 (gamma Leu-373-Pro-489). The digests were separated by reverse-phase high-performance liquid chromatography and labeled peptides identified by amino-terminal sequence analysis. [I-125]Azido-cholesterol labeling was localized to peptides that contain almost exclusively the alpha-M4, alpha-M1 and gamma-M4 membrane spanning segments. These results establish that the binding domain for cholesterol is at the lipid-protein interface of the AChR. (C) 1998 Elsevier Science B.V. All rights reserved.