Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.
Catch-and-hold activation of muscle acetylcholine receptors having transmitter binding site mutations.
复制标题
捕获并保持激活具有递质结合位点突变的肌肉乙酰胆碱受体。
DOI：
10.1016/j.bpj.2014.04.057
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发表时间：
2014
影响因子：
3.4
通讯作者：
Auerbach,Anthony
中科院分区：
文献类型：
--
作者：
Purohit,Prasad;Bruhova,Iva;Gupta,Shaweta;Auerbach,Anthony
激动剂打开受体，因为它们的靶位点在蛋白质的活性构象中比静息构象具有更高的亲和力。我们使用单通道电生理学测量低亲和力（LA）和高亲和力（HA）的平衡解离常数乙酰胆碱在成人型肌肉小鼠烟碱受体（AChRs）激动剂结合位点氨基酸突变。对于一系列激动剂和α Y 93、αG147、αW149、αY190、αY198、εW55和δW57的所有突变，LA结合能的变化约为HA结合能的一半。结果分析为LA和HA激动剂结合之间的线性自由能关系，其斜率（κ）给出了LA复合物建立的总结合化学势的分数。LA和HA结合能之间的线性相关性表明，整个结合过程是通过一个集成的机制（捕获和保持）。对于激动剂和上述突变，κ = 0.5，但两个残基的侧链取代具有显著更高（0.90;αG153）或更低（0.25;εP121）的斜率。结果表明，环B、环C和非α表面中的骨架重排参与LA结合和LA ParticipHA亲和力转换。看来，AChR激活的所有中间步骤包括一个单一的，能量耦合的过程。
Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations ofαY93,αG147,αW149,αY190,αY198,εW55, andδW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations,κ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90;αG153) or lower (0.25;εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-αsurface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.