Agonist binding and affinity state transitions in reconstituted nicotinic acetylcholine receptors revealed by single and sequential mixing stopped-flow fluorescence spectroscopies.
Agonist binding and affinity state transitions in reconstituted nicotinic acetylcholine receptors revealed by single and sequential mixing stopped-flow fluorescence spectroscopies.
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通过单一和连续混合停流荧光光谱揭示重建烟碱乙酰胆碱受体中的激动剂结合和亲和状态转变。
DOI：
10.1016/s0005-2736(98)00133-3
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Krishnan,NS
中科院分区：
文献类型：
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作者：
Raines,DE;Krishnan,NS
用单次和顺序混合停流荧光光谱法测定了烟碱乙酰胆碱受体（nAcChoRs）重组为二油基磷脂酰胆碱（DOPC）或二油基磷脂酰胆碱、二油基磷脂酸和胆固醇的混合物（DOPC/DOPA/胆固醇）的亲和状态。这些技术具有毫秒级的时间分辨率，允许在与荧光部分激动剂Dns-C6-Cho混合后，根据其特征的Dns-C6-Cho解离率来分辨nAcChoR的低和高亲和构象状态。我们的研究表明，在激动剂诱导的亲和状态转换之前，重构成DOPC或DOPC/DOPA/胆固醇的nAcChoRs主要处于对激动剂具有低亲和力的构象状态。长时间暴露于Dns-C6-Cho中，几乎所有DOPC/DOPA/胆固醇重组的nAcChoRs都转化为高亲和力状态。相比之下，Dns-C6-Cho仅将所有dopc重构的nAcChoRs的一半转化为高亲和状态。另一半则保持在低亲和力状态，对Dns-C6-Cho的kd值为0.61±0.07 μM。这与先前报道的Dns-C6-Cho与天然膜中低亲和力，静息状态nAcChoRs结合的kdi相似。然而，通过将DOPC重组的nAcChoRs重新重组成由DOPC/DOPA/胆固醇组成的双分子层，可以促进其亲和状态的转换。这些结果表明，脂质双分子层组成调节了nAcChoR激动剂诱导的亲和状态转变。
The affinity state of nicotinic acetylcholine receptors (nAcChoRs) reconstituted into either dioleoylphosphatidylcholine (DOPC) or a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidic acid, and cholesterol (DOPC/DOPA/cholesterol) has been determined using single and sequential mixing stopped-flow fluorescence spectroscopies. These techniques have millisecond temporal resolution, permitting low- and high-affinity conformational states of the nAcChoR to be resolved following mixing with the fluorescent partial agonist Dns-C6-Cho from their characteristic Dns-C6-Cho dissociation rates. Our studies reveal that prior to agonist-induced affinity state conversion, nAcChoRs reconstituted into either DOPC or DOPC/DOPA/cholesterol are predominantly in a conformational state that has a low affinity for agonist. Prolonged exposure to Dns-C6-Cho converts nearly all DOPC/DOPA/cholesterol-reconstituted nAcChoRs to the high-affinity state. In contrast, Dns-C6-Cho converts only half of all DOPC-reconstituted nAcChoRs to the high-affinity state. The other half persists in a low-affinity state characterized by a Kdfor Dns-C6-Cho of 0.61±0.07 μM. This Kdis similar to that previously reported for Dns-C6-Cho binding to low-affinity, resting-state nAcChoRs in native membranes. However, affinity state conversion of DOPC-reconstituted nAcChoRs may be facilitated by re-reconstituting them into bilayers composed of DOPC/DOPA/cholesterol. These results indicate that the lipid bilayer composition modulates nAcChoR agonist-induced affinity state transitions.