Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, 9160000, Chile.
Millennium Nucleus of Ion Channel-Associated Diseases, Santiago, 9160000, Chile.
J Neurosci. 2021 Oct 13;41(41):8475-8493. doi: 10.1523/JNEUROSCI.0345-21.2021. Epub 2021 Aug 26.
In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons. Post-translational modifications are one of the main molecular mechanisms involved in adjusting the sensitivity of sensory ion channels to changing environmental conditions. Here we show, for the first time, that constitutive phosphorylation of the well-conserved serine 29 within the N-terminal domain negatively modulates TRPM8 channel activity, reducing its activation by agonists and decreasing the number of active channels at the plasma membrane. Basal phosphorylation of TRPM8 acts as a key regulator of its function as the main cold-transduction channel, significantly contributing to the net response of primary sensory neurons to temperature reductions. This reversible and dynamic modulatory mechanism opens new opportunities to regulate TRPM8 function in pathologic conditions where this thermo-TRP channel plays a critical role.
在哺乳动物中，外周冷热敏神经元进行的环境冷感主要依赖于 TRPM8，这是一种已经进化成为主要分子冷传感器的离子通道。该 TRP 通道受冷、冷却化合物（如薄荷醇）、电压和渗透压升高的激活。TRPM8 的功能受激酶活性调节，在静息状态下，该激酶活性使通道磷酸化。然而，哪些特定的残基、这种翻译后修饰如何调节 TRPM8 的活性，以及其对冷感的影响仍然知之甚少。通过质谱分析，我们在小鼠同源物中鉴定出 N 端的四个丝氨酸残基（S26、S29、S541 和 S542）在静息状态下持续磷酸化。通过钙成像和膜片钳记录来检查 TRPM8 的功能，结果表明，用蛋白激酶抑制剂 staurosporine 处理可增强其对冷和薄荷醇的反应。S29A 突变足以增加 TRPM8 的活性，表明该残基的磷酸化是这种负调节的主要分子决定因素。生物物理和全内反射荧光分析揭示了未磷酸化的 TRPM8 增强反应的双重机制：电压激活曲线向更负的电位移动，以及质膜上活性通道的数量增加。重要的是，基础激酶活性负调节雄性和雌性小鼠冷热敏感受器中的 TRPM8 功能，这一观察结果可以通过数学模型来解释。总的来说，我们的研究结果表明，通过控制 TRPM8 的基础磷酸化状态，可以快速和可逆地微调冷温度检测，这是一种作为初级感觉神经元中这种热 TRP 通道功能的动态分子制动器的机制。翻译后修饰是调节感觉离子通道对环境条件变化的敏感性的主要分子机制之一。在这里，我们首次表明，N 端保守丝氨酸 29 的组成性磷酸化负调节 TRPM8 通道活性，降低其激动剂的激活作用，并减少质膜上的活性通道数量。TRPM8 的基础磷酸化作为其功能的关键调节剂，作为主要的冷转导通道，显著促进初级感觉神经元对温度降低的净反应。这种可逆和动态的调节机制为在这种热 TRP 通道发挥关键作用的病理条件下调节 TRPM8 的功能开辟了新的机会。
