Pertusa María, González Alejandro, Hardy Paulina, Madrid Rodolfo, Viana Félix
From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and the Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, 03550 Alicante, Spain
From the Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, 9160000 Santiago, Chile and.
J Biol Chem. 2014 Aug 8;289(32):21828-43. doi: 10.1074/jbc.M114.565994. Epub 2014 Jun 10.
TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40-60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.
瞬时受体电位香草酸亚型8（TRPM8）是一种由寒冷、电压以及薄荷醇等冷却化合物激活的非选择性阳离子通道，是躯体感觉系统初级感觉神经元中寒冷温度的主要分子探测器。TRPM8的N端结构域由693个氨基酸组成，但对其对通道功能的贡献了解甚少。在这里，我们在TRPM8的初始N端内鉴定出两个不同区域，它们对通道活性以及正确的折叠和组装有不同的贡献。缺失或替换前40个残基会产生对寒冷和薄荷醇反应增强的通道。这些突变体的热激活阈值向更高温度偏移了2°C，薄荷醇剂量反应曲线向更低浓度偏移。定点诱变筛选显示，脯氨酸在Ser-26或Ser-27位点的单点突变导致对寒冷和薄荷醇的反应有类似的增加。对S27P突变体的电生理分析表明，对激动剂的敏感性增强与激活电压依赖性的向左偏移有关，增加了在生理膜电位下通道开放的概率。此外，我们发现包含40-60位的区域是TRPM8正确折叠和组装的关键要素。该区域内不同的缺失和突变导致通道功能受损，并保留在内质网中。我们的结果表明，TRPM8 N端结构域的初始区域对热和化学敏感性以及这种多模态离子通道的正确生物合成有至关重要的贡献。
