Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.
Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.
复制标题
通过使用顺序混合停流荧光光谱法解析了与鱼雷突触后膜结合的瞬时低亲和力激动剂。
DOI：
10.1021/bi971689w
复制
发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
Krishnan,NS
中科院分区：
文献类型：
--
作者：
Raines,DE;Krishnan,NS
我们使用顺序混合停流荧光光谱检测了荧光激动剂 Dns-C6-Cho 与烟碱乙酰胆碱受体 (nAcChoR) 的低亲和力和高亲和力状态的结合。我们解决低亲和力和高亲和力结合的方法是首先将受体膜与荧光部分激动剂 Dns-C6-Cho 预孵育 15 毫秒至 1000 秒，然后在化学稀释到过量乙酰胆碱中时跟踪荧光衰减。快速和慢速衰减反映了 Dns-C6-Cho 与低亲和力和高亲和力受体的解离，其速率分别为 140 ± 27 s-1 和 0.1 ± 0.02 s-1。随着预孵育时间的增加，低亲和力受体的数量减少，而高亲和力受体的数量以 Dns-C6-Cho 浓度依赖性方式增加，这与激动剂诱导的亲和状态转换的当前模型一致。在 Dns-C6-Cho 的受体激活浓度下，高亲和力受体形成的表观速率近似于离子流脱敏状态的表观速率，这意味着快速脱敏状态的激动剂解离速率与平衡慢速脱敏状态无法区分。低亲和力结合位点的 KD 被确定为 1.1 μM，根据 Dns-C6-Cho 浓度快速衰减幅度的增加，预温育时间足够短以最小化亲和状态转换。假设双分子结合率为 108M-1s-1，则对低亲和力结合进行第二次估计为 1.4 μM。我们还检测到与 Dns-C6-Cho 抑制的 nAcChoR 构象异构化一致的荧光增强。
We have detected the binding of the fluorescent agonist Dns-C6-Cho to both low- and high-affinity states of the nicotinic acetylcholine receptor (nAcChoR) using sequential mixing stopped-flow fluorescence spectroscopy. Our approach to resolving low- and high-affinity binding was to first preincubate receptor membranes with the fluorescent partial agonist Dns-C6-Cho for 15 ms to 1000 s and then to follow the fluorescence decay upon chemical dilution into excess acetylcholine. The fast and slow decays, reflecting Dns-C6-Cho dissociation from low- and high-affinity receptors, had rates of 140 ± 27 s-1and 0.1 ± 0.02 s-1, respectively. With increasing preincubation times, the number of low-affinity receptors decreased while the number of high-affinity receptors increased in a Dns-C6-Cho concentration-dependent manner consistent with current models for agonist-induced affinity state conversion. At receptor-activating concentrations of Dns-C6-Cho, the apparent rates with which high-affinity receptors formed approximated those of ion flux desensitization, implying that the fast desensitized state has an agonist dissociation rate that is indistinguishable from the equilibrium slow desensitized state. TheKDfor the low-affinity binding site was determined to be 1.1 μM from the increase in the amplitude of the fast decay with Dns-C6-Cho concentration with preincubation times that were sufficiently brief to minimize affinity state conversion. Assuming a bimolecular association rate of 108M-1s-1, a second estimate of 1.4 μM was made for low-affinity binding. We also detected a fluorescence enhancement consistent with a conformational isomerization of Dns-C6-Cho-inhibited nAcChoRs.