Luo Yuhuan, Chen Shijia, Wu Fei, Jiang Chunming, Fang Marong
Department of Pediatrics, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
National Clinical Research Center for Child Health, Children's Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Front Aging Neurosci. 2022 Oct 24;14:1033434. doi: 10.3389/fnagi.2022.1033434. eCollection 2022.
Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, is involved in many physiological and pathological processes, including temperature sensing, synaptic plasticity regulation, and neurodegenerative diseases. However, the gating mechanism of TRPM2 channel is complex, which hinders its functional research. With the discovery of the Ca binding site in the S2-S3 domain of TRPM2 channel, more and more attention has been drawn to the role of the transmembrane segments in channel gating. In this study, we focused on the D820-F867 segment around the S2 domain, and identified the key residues on it. Functional assays of the deletion mutants displayed that the deletions of D820-W835 and L836-P851 destroyed channel function totally, indicating the importance of these two segments. Sequence alignments on them found three polar and charged residues with high conservation (D820, E829, and R845). D820A, E829A, and R845A which removed the charge and the side chain of the residues were tested by 500 μM adenosine diphosphate-ribose (ADPR) or 50 mM Ca. E829A and R845A affected the characteristic of channel currents, while D820A behaved similarly to WT, indicating the participations of E829 and R845 in channel gating. The charge reversing mutants, E829K and R845D were then constructed and the electrophysiological tests showed that E829A and E829K made the channel lose function. Interestingly, R845A and R845D exhibited an inactivation process when using 500 μM ADPR, but activated normally by 50 mM Ca. Our data suggested that the negative charge at E829 took a vital part in channel activation, and R845 increased the stability of the Ca combination in S2-S3 domain, thus guaranteeing the opening of TRPM2 channel. In summary, our identification of the key residues E829 and R845 in the transmembrane segments of TRPM2. By exploring the gating process of TRPM2 channel, our work helps us better understand the mechanism of TRPM2 as a potential biomarker in neurodegenerative diseases, and provides a new approach for the prediction, diagnosis, and prognosis of neurodegenerative diseases.
瞬时受体电位香草酸亚家族成员2（TRPM2）是一种非选择性阳离子通道，参与许多生理和病理过程，包括温度感知、突触可塑性调节以及神经退行性疾病。然而，TRPM2通道的门控机制复杂，这阻碍了其功能研究。随着TRPM2通道S2 - S3结构域中钙结合位点的发现，跨膜片段在通道门控中的作用越来越受到关注。在本研究中，我们聚焦于S2结构域周围的D820 - F867片段，并确定了其上的关键残基。缺失突变体的功能测定表明，D820 - W835和L836 - P851的缺失完全破坏了通道功能，表明这两个片段的重要性。对它们进行序列比对发现三个具有高度保守性的极性带电残基（D820、E829和R845）。通过500μM二磷酸腺苷核糖（ADPR）或50mM钙对去除残基电荷和侧链的D820A、E829A和R845A进行测试。E829A和R845A影响通道电流特性，而D820A表现与野生型相似，表明E829和R845参与通道门控。随后构建了电荷反转突变体E829K和R845D，电生理测试表明E829A和E829K使通道失去功能。有趣的是，当使用500μM ADPR时，R845A和R845D表现出失活过程，但在50mM钙作用下正常激活。我们的数据表明，E829处的负电荷在通道激活中起关键作用，R845增加了S2 - S3结构域中钙结合的稳定性，从而保证了TRPM2通道的开放。总之，我们鉴定了TRPM2跨膜片段中的关键残基E829和R845。通过探索TRPM2通道的门控过程，我们的工作有助于我们更好地理解TRPM2作为神经退行性疾病潜在生物标志物的机制，并为神经退行性疾病的预测、诊断和预后提供了新方法。
