Functional analysis of the channel enzyme TRPM2 in vitro and in vivo : species-specific role of the NUDT9H domain and phenotypic characterization of a TRPM2 knockout of the model organism Nematostella vectensis

  • Funktionale Analyse des Kanalenzym TRPM2 in vitro und in vivo : Speziesspezifische Rolle der NUDT9H Domäne und phänotypische Charakterisierung eines TRPM2 Knockout des Modelorganismus Nematostella vectensis

Ehrlich, Wiebke; Kühn, Frank (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


The human ortholog of the Ca2+ permeable cation channel TRPM2 (hsTRPM2) is critically involved in oxidative stress-induced apoptosis which implicates participation in several important pathophysiological processes. The comparative analysis of different vertebrate and invertebrate TRPM2 species variants revealed valuable insights into the structure-function relationship of TRPM2. The presence of a C-terminal domain (NUDT9H), derived from the human ADP-ribose diphosphatase NUDT9, represents a unique feature of TRPM2 and is crucial for channel gating, at least in vertebrate orthologs. For a long time, NUDT9H was thought to function as only binding site for the principal TRPM2 agonist ADP-ribose (ADPR). However, investigations on the TRPM2 ortholog of the sea anemone Nematostella vectensis strongly suggest the existence of an additional and novel ADPR binding site in the N-terminus of TRPM2. Meanwhile, this N-terminal ADPR binding site was identified in a species variant of zebrafish (Danio rerio) as well as in human TRPM2 using cryo-electron microscopy analysis.At the beginning of this work, it was generally accepted that the functional role of the NUDT9H domain is strikingly different between vertebrate and invertebrate variants of TRPM2. However, neither the general importance of the N-terminal ADPR binding site nor the question which factors determine the species-specific role of the NUDT9H domain were reasonably understood. Moreover, the intriguing question about the physiological function of this important mammalian ion channel in invertebrates like the cnidarian model organism Nematostella vectensis, had not been addressed until then.The present study aimed to produce valuable contributions to our understanding of these important questions. In the first part of this thesis the species-specific role of NUDT9H and N-terminal ADPR binding site was examined by functional in-vitro analysis of diverse channel mutants as well as TRPM2 chimeras. In addition, co-immunoprecipitation (Co-IP) experiments were performed as a novel approach to detect structural interactions between channel and NUDT9H domain. The second part of this work covers the phenotypical characterization of a TRPM2 knockout in vivo (Nematostella vectensis), with special emphasis on conditions of environmental stress, i.e., exposure to reactive oxygen species or high temperature. In this way it should be tested whether the physiological role of TRPM2 in cnidarians is similar to that in mammals or whether it has changed significantly in the course of metazoan evolution. The data of this thesis reveal that the N-terminal ADPR binding site is equally crucial for ADPR-dependent channel gating in the far distantly related TRPM2 orthologs of human, zebrafish, and sea anemone, as well as for channel activation induced by 2-Aminoethoxydiphenyl borate. Furthermore, it was shown that the NUDT9H domains of hsTRPM2 and drTRPM2 can be swapped producing functional channels. This work also confirmed the crucial importance of a highly conserved asparagine residue within the NUDT9H domain for channel function of vertebrate TRPM2 orthologs, while in nvTRPM2 it only affects the sensitivity to oxidative stress. The Co-IP data demonstrate that at least in hsTRPM2 this asparagine residue is critically involved in the structural interactions between NUDT9H and channel domain. Strikingly, the in-vivo analyses of a TRPM2 knockout of Nematostella vectensis revealed that in sea anemones this ion channel is involved both, in the cellular response to oxidative stress and to high temperature, and therefore exerts a similar physiological function as in humans. As a side result, strong expression of an ortholog of the human ADPR-pyrophoshatase NUDT9 was detected in Nematostella vectensis. The findings of this thesis may serve as the basis for further investigations in order to better understand the functional evolution of this ion channel. Future studies on the well-established model organism Nematostella vectensis could help to define the physiological spectrum of TRPM2 and to better understand the function and regulation of the intracellular ADPR level.