Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site.
Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site.
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通过半胱氨酸扫描诱变探测烟碱乙酰胆碱受体的激动剂结构域，揭示了靠近 α-银环蛇毒素结合位点的残基。
DOI：
10.1021/bi982656z
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Hawrot,E
中科院分区：
文献类型：
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作者：
Spura,A;Russin,TS;Freedman,ND;Grant,M;McLaughlin,JT;Hawrot,E
我们构建了一系列半胱氨酸替代突变体，以鉴定小鼠肌肉烟碱乙酰胆碱受体（AChR）中参与α-班加罗毒素（α-Bgtx）结合的残基。在HEK 293衍生的TSA-201细胞中短暂表达后，用巯基特异性试剂对引入的半胱氨酸进行共价修饰，发现α亚基残基W187、V188、F189、Y190和P194是溶剂可及的，并且可以在天然受体中参与α- bgtx结合位点。完整受体的这些结果与α-Bgtx/受体-十二肽复合物的核磁共振研究一致[Basus， V., Song]。陈晓明，陈晓明，陈晓明，等（1993）生物化学[j]。我们对F189C突变体进行了更详细的分析，因为该位点在结合Bgtx的achr和不结合Bgtx的某些神经元achr之间存在很大差异。在饱和条件下，用溴乙酰胆碱（BrACh）或[2-（三甲基铵）乙基]甲烷-硫代磺酸盐（MTSET）处理表达F189C的完整细胞，结果α-Bgtx结合位点的数量显著减少（~ 55−70%）。位点的减少似乎对α/γ和α/δ位点的影响程度相同，α w187c和α f189c是这一问题研究的两个突变体。与MTSET和BrACh相比，缺乏烷基胺实体的试剂，如甲基乙硫磺酸盐（MMTS）、带负电荷的2-磺基乙基甲烷硫磺酸盐（MTSES）或带正电荷的氨基乙基甲基硫磺酸盐（MTSEA），对α-Bgtx与αW187C、αV188C或αF189C突变体受体的最大结合影响很小或没有影响。惊人的烷基铵依赖性表明，拴系修饰基团与激动剂结合域内的负亚位点的相互作用是观察到的毒素结合阻断的主要原因。
We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in α-bungarotoxin (α-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that α subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the α-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an α-Bgtx/receptor−dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993)Biochemistry32, 12290−12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease (∼55−70%) in the number of α-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both α/γ and α/δ sites to the same extent, as shown for αW187C and αF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of α-Bgtx to the αW187C, αV188C, or αF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.