Functional analyis of an early evolutionary ancestor of cation channels of the TRPM-subfamily from Nematostella vectensis in vitro and in vivo.

本项目的目的是阐明人类TRPM2通道的复杂结构-功能关系。为此，我们对一个与海葵有较远亲缘关系的变种进行了功能鉴定。选择这种不同寻常的实验方法有两个原因。首先，hTRPM2和nvTRPM2之间的序列同源性只有31%左右，这应该有助于识别TRPM2的通道门控所必需的结构。此外，从进化的角度来看，我们想要找出这两个同源基因的生理功能在多大程度上仍然是一致的。到目前为止，我们所获得的结果大大有助于改变传统的依赖ADPR激活TRPM2通道的模式。基于我们的实验数据，我们能够推断出存在一个新的、NUDT9H非依赖的ADPR结合口袋，同时这一点已经被TRPM2的几个结构分析所证实。此外，我们对NUDT9H结构域的可变功能作用也有了新的认识。我们还首次进行了N端ADPR结合位点的药理学表征，并对不同物种的NUDT9H结构域的各种通道嵌合体进行了功能分析。除了其他几个小组获得的结构数据外，我们的实验结果还有助于更好地理解TRPM2的结构-功能关系。然而，NUDT9H结构域在脊椎动物TRPM2通道激活中的功能作用远未得到最终阐明。同样，关于统一的N-末端ADPR结合位点的许多问题仍然没有回答，例如它对2-APB激活通道的额外影响，或者观察到一些物种变体表现出相反的底物特异性。我们希望在前线做出贡献的另一个同样重要的问题是nvTRPM2在体内的生理功能的表征。我们现在已经完成了广泛和耗时的准备工作，并准备开始调查。TRPM2的N-末端ADPR结合口袋属于核苷酸结合域的超家族的认识也开辟了新的视角，例如在原核生物中寻找TRPM2的进化前体或鉴定新的、以前未被识别的TRPM通道的相互作用伙伴。拟议项目的计划实验以及我们合作伙伴的持续支持将为破译TRPM2的门控机制和生理功能做出重要贡献。

The aim of this project is to elucidate the complex structure-function relationship of the human TRPM2 channel. For this purpose, a far distantly related species variant from the sea anemone Nematostella vectensis was functionally characterized. This unusual experimental approach was selected for two reasons. First, the sequence homology between hTRPM2 and nvTRPM2 is only about 31%, which should help to identify the structures that are essential for channel gating of TRPM2. Furthermore, from an evolutionary point of view we want to find out to what extent the physiological function of these two orthologues are still congruent.The results we have obtained so far has contributed significantly to a paradigm shift in the conventional model of the ADPR-dependent activation of TRPM2 channels. Based on our experimental data we were able to deduce the existence of a novel, NUDT9H-independent ADPR binding pocket, which in the meantime has been verified by several structural analyzes of TRPM2. In addition, we initiated the new understanding of the variable functional role of the NUDT9H domain. We also performed a first pharmacological characterization of the N-terminal ADPR binding site and functionally analyzed various channel chimeras with NUDT9H domains from different species. In addition to the structural data obtained by several other groups, our experimental findings has contributed significantly to a better understanding of the structure-function relationship of TRPM2.Nevertheless, the functional role of the NUDT9H domain for the activation of the vertebrate TRPM2 channels is far from being conclusively clarified. Likewise, many questions remain unanswered regarding the uniform N-terminal ADPR binding site, e.g. its additional impact on channel activation by 2-APB or the observation that some species variants show opposite substrate specificities. An equally important question where we wish to contribute at the front line is the characterization of the physiological function of nvTRPM2 in vivo. We have now completed the extensive and time consuming preparations and are ready to start our investigations. The realization that the N-terminal ADPR binding pocket of TRPM2 belongs to a superfamily of nucleotide binding domains also opens up new perspectives e. g. the search for evolutionary precursors of TRPM2 in prokaryotes or the identification of new, previously unrecognized interaction partners of TRPM channels. The planned experiments of the proposed project together with the ongoing support of our cooperation partners will provide important contributions to decipher the gating mechanism and physiological function of TRPM2.