瞬时受体电位美司他丁2(TRPM2),非选择性阳离子通道,参与许多生理和病理过程,包括温度传感,突触可塑性调节,和神经退行性疾病。然而,TRPM2通道的门控机制复杂,这阻碍了它的功能研究。随着TRPM2通道S2-S3结构域中Ca2+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重视。在这项研究中,我们专注于S2域周围的D820-F867段,并确定了其上的关键残留物。缺失突变体的功能分析显示,D820-W835和L836-P851的缺失完全破坏了通道功能,说明了这两个部分的重要性。对它们的序列比对发现了三个具有高保守性的极性和带电残基(D820、E829和R845)。D820A,E829A,用500μM二磷酸腺苷-核糖(ADPR)或50mMCa2+测试除去电荷和残基侧链的R845A。E829A和R845A影响了通道电流的特性,而D820A的行为与WT相似,指示E829和R845在通道门控中的参与。电荷逆转突变体,然后构建E829K和R845D,电生理测试表明E829A和E829K使通道失去功能。有趣的是,R845A和R845D在使用500μMADPR时表现出失活过程,但通常被50mMCa2+激活。我们的数据表明,E829的负电荷在通道激活中起着至关重要的作用,和R845增加了S2-S3结构域中Ca2+组合的稳定性,从而保证TRPM2通道的开通。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了解TRPM2作为神经退行性疾病的潜在生物标志物的机制,并为预测提供了一种新的方法,诊断,和神经退行性疾病的预后。
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 Ca2+ 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 Ca2+. 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 Ca2+. Our data suggested that the negative charge at E829 took a vital part in channel activation, and R845 increased the stability of the Ca2+ 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.