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

本项目旨在阐明人体TRPM2通道复杂的结构-功能关系。为此，对海葵Nematostella vectensis的一个远亲物种变异进行了功能表征。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通道门控所必需的结构。此外，从进化的角度来看，我们想找出这两个同源物的生理功能在多大程度上仍然是一致的。到目前为止，我们获得的结果对传统的adpr依赖性TRPM2通道激活模型的范式转变做出了重大贡献。根据我们的实验数据，我们能够推断出一种新的，不依赖于nudt9h的ADPR结合口袋的存在，同时通过对TRPM2的多次结构分析证实了这一点。此外，我们对NUDT9H结构域的可变功能作用有了新的认识。我们还首次对n端ADPR结合位点进行了药理学表征，并对来自不同物种的NUDT9H结构域的各种通道嵌合体进行了功能分析。除了其他几个研究组获得的结构数据外，我们的实验发现对更好地理解TRPM2的结构-功能关系有重要贡献。然而，NUDT9H结构域在激活脊椎动物TRPM2通道中的功能作用还远未明确。同样，关于统一的n端ADPR结合位点的许多问题仍未得到解答，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异表现出相反的底物特异性。我们希望在前线贡献的一个同样重要的问题是nvTRPM2在体内生理功能的表征。我们现在已经完成了广泛而耗时的准备工作，准备开始调查。认识到TRPM2的n端ADPR结合口袋属于核苷酸结合域的超家族，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。本项目的实验计划以及合作伙伴的持续支持将为破解TRPM2的门控机制和生理功能提供重要贡献。

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.