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

Nematostella vectensis TRPM 亚科阳离子通道早期进化祖先的体外和体内功能分析。

• 批准号: 269020709
• 负责人: Privatdozent Dr. Frank J. P. Kühn
• 金额: --
• 依托单位: Institut für Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/269020709?language=en
• 关键词: Functional; analyis; early; evolutionary; ancestor

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.

本项目的目的是阐明人类TRPM 2通道的复杂结构-功能关系。为此目的，一个遥远的相关物种变异的海葵Nematostella vectensis的功能特点。选择这种不寻常的实验方法有两个原因。首先，hTRPM 2和nvTRPM 2之间的序列同源性仅约为31%，这应该有助于识别TRPM 2通道门控所必需的结构。此外，从进化的角度来看，我们想找出在何种程度上的生理功能，这两个直系同源物仍然congruent.The结果，我们迄今为止已经获得了显着的ADPR依赖激活TRPM 2通道的传统模型的范式转变作出了贡献。基于我们的实验数据，我们能够推断出一个新的，NUDT 9 H-独立的ADPR结合口袋的存在，这在同一时间已被TRPM 2的几个结构分析所验证。此外，我们还对NUDT 9 H结构域的可变功能作用有了新的认识。我们还进行了N-末端ADPR结合位点的第一次药理学表征，并对来自不同物种的具有NUDT 9 H结构域的各种通道嵌合体进行了功能分析。除了其他几个研究小组获得的结构数据外，我们的实验结果为更好地理解TRPM 2的结构-功能关系做出了重要贡献。然而，NUDT 9 H结构域在脊椎动物TRPM 2通道激活中的功能作用远未得到明确的阐明。同样，关于均匀的N-末端ADPR结合位点的许多问题仍然没有答案，例如其对2-APB通道激活的额外影响或观察到一些物种变体显示相反的底物特异性。我们希望在前线做出贡献的一个同样重要的问题是nvTRPM 2在体内生理功能的表征。我们现在已经完成了大量和耗时的准备工作，并准备开始调查。TRPM 2的N端ADPR结合口袋属于核苷酸结合结构域超家族的认识也开辟了新的视角，例如：G.在原核生物中寻找TRPM 2的进化前体或识别新的、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TRPM 2的门控机制和生理功能做出重要贡献。