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

• Applicant: Privatdozent Dr. Frank J. P. Kühn
• Subject Area: Anatomy and Physiology ; Biophysics ; Cell Biology
• Term: from 2014 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269020709

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.

• DFG Programme: Research Grants