Microscopic kinetics and energetics distinguish GABAA receptor agonists from antagonists
Microscopic kinetics and energetics distinguish GABAA receptor agonists from antagonists
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DOI：
10.1016/s0006-3495(01)75909-7
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发表时间：
2001-11-01
影响因子：
3.4
通讯作者：
Westbrook, GL
中科院分区：
文献类型：
--
作者：
Jones, MV;Jonas, P;Westbrook, GL
虽然激动剂和竞争性拮抗剂可能占据配体门控通道上的重叠结合位点，但这些相互作用不可能相同，因为激动剂导致通道开放，而拮抗剂则不会。一种解释是，只有激动剂结合对受体执行足够的工作，以引起导致门控的构象变化。GABAA和烟碱乙酰胆碱受体的激动剂结合速率比扩散限制过程的预期慢，这表明激动剂结合涉及能量需求事件，从而支持了这一观点。这一假说预测竞争性拮抗剂结合应该比激动剂结合需要更少的活化能。为了验证这一想法，我们开发了一种新的基于电子卷积的方法来比较GABAA受体激动剂和拮抗剂在大鼠海马神经元外贴中的结合和非结合动力学。激动剂和拮抗剂的解结合率与亲和力急剧相关。与激动剂不同，测试的四种拮抗剂中有三种的结合速率快，不依赖于亲和力，并且可以通过扩散和脱水限制过程来解释。相反，激动剂结合涉及额外的能量需要的步骤，与通道门控是由激动剂触发的配体结合位点内的运动启动的想法一致。拮抗剂结合似乎不会产生这种运动，事实上可能会阻止它们。
Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABAA and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel eleconvolution-based method to compare binding and unbinding kinetics of GABAA receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.