Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes
Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes
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DOI：
10.1073/pnas.0603225103
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发表时间：
2006-07-05
影响因子：
11.1
通讯作者：
Jayaraman, Vasanthi
中科院分区：
文献类型：
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作者：
Ramanoudjame, Gomathi;Du, Mei;Jayaraman, Vasanthi
Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic酸(AMPA)受体是哺乳动物中枢快速兴奋性突触传递的主要介质。细胞外配体结合域的结构表明，配体结合域中裂隙的闭合程度控制着受体的激活程度。在这里，我们开发了一种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结构域中裂隙闭合的程度。这些研究表明，野生型蛋白呈现出与激活程度相关的分级裂隙闭合，这与晶体结构定性一致。然而，在载脂蛋白和激动剂结合状态之间的裂隙闭合程度的变化比在晶体结构中观察到的要小。我们还用这种方法研究了L650T突变体，表明在溶液中，该突变体的α-氨基-5-甲基-3-羟基-4-异恶唑丙酸酯结合形式主要以一种比基于活性预测的更紧密的构象存在，表明裂隙闭合程度不能单独用作受体激活程度的衡量标准，除了裂隙闭合之外，可能还有其他机制调节给定激动剂激活程度的微妙程度。
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.