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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes
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Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

机译：AMPA受体的变构机制：基于FRET的构象变化研究

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摘要

alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.
机译：α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体是哺乳动物CNS中快速兴奋性突触传递的主要介质。细胞外配体结合结构域的结构表明，配体结合结构域中的裂缝闭合程度控制受体的活化程度。在这里，我们开发了基于荧光共振能量转移的探针，使我们能够研究溶液中分离的配体结合域中的裂缝闭合程度。这些研究表明，野生型蛋白表现出与活化程度相关的分级裂隙闭合，其与晶体结构在质量上是一致的。但是，载脂蛋白和激动剂结合状态之间的裂缝闭合程度的变化小于晶体结构中观察到的变化。我们还使用这种方法研究了L650T突变体，并表明在溶液中该突变体的α-氨基-5-甲基-3-羟基-3-羟基-4-异恶唑丙酸酯结合形式主要存在于比预期更封闭的构象中基于该活性，表明不能单独使用closure裂闭合度作为受体激活程度的量度，并且除了c裂闭合以外，可能还有其他机制介导给定激动剂激活程度的细微差别。 。

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