High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol
High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol
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DOI：
10.1038/nn1665
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发表时间：
2006-04-01
影响因子：
25
通讯作者：
Patapoutian, A
中科院分区：
文献类型：
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作者：
Bandell, M;Dubin, AE;Patapoutian, A
薄荷脑是一种从薄荷叶中提取的清凉化合物，广泛用作调味化学品。Menglutamine激活瞬时受体电位melastatin 8（TRPM 8），这是一种也由冷、电压和磷脂酰肌醇-4，5-二磷酸（PIP 2）激活的离子通道。在这里，我们研究了薄荷醇激活小鼠TRPM 8的机制。使用一种新的高通量方法，我们筛选了一个随机突变体库，该库由类似于14，000个单独的TRPM 8突变体组成，以获得在保留通道功能的同时对薄荷醇的反应受到影响的克隆。我们确定了两个区域的薄荷醇敏感性的决定因素：假定的跨膜段2（S2）和C-末端TRP结构域。对这些突变体的分析表明，薄荷醇的激活涉及与冷、电压或PIP 2门控不同且可分离的门控机制。值得注意的是，TRP结构域突变主要减弱薄荷醇的功效，表明该结构域影响初始结合下游的事件。相反，S2突变强烈地改变了薄荷醇激活的浓度依赖性，提高了S2影响薄荷醇结合的可能性。
Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of similar to 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.