ADP-Ribose Activates the TRPM2 Channel from the Sea Anemone Nematostella vectensis Independently of the NUDT9H Domain

The human redox-sensitive Transient receptor potential melastatin type 2 ( h TRPM2) channel contains the C-terminal Nudix hydrolase domain NUDT9H which most likely binds ADP-ribose. During oxidative stress, the intracellular release of ADP-ribose triggers the activation of h TRPM2. The TRPM2 orthologue from Nematostella vectensis ( nv ) is also stimulated by ADP-ribose but not by the oxidant hydrogen peroxide. For further clarification of the structure-function relationships of these two distantly related channel orthologues, we performed whole-cell as well as single channel patch-clamp recordings, Ca 2+ -imaging and Western blot analysis after heterologous expression of wild-type and mutated channels in HEK-293 cells. We demonstrate that the removal of the entire NUDT9H domain does not disturb the response of nv TRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nv TRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nv TRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original h NUDT9 enzyme. Whenever the enzyme domain was mutated to presumably inactive variants, channel activation by hydrogen peroxide could be achieved. Moreover, we found strong evidences for ADPRase activity of the isolated NUDT9H domain of nv TRPM2 in co-expression experiments with the C-terminally truncated nv TRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nv TRPM2 to respond to oxidative stress. In striking contrast, the channel function of the h TRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nv TRPM2 as a channel gated by ADP-ribose through a novel mechanism. We conclude that the endogenous NUDT9H domain does not directly affect ADP-ribose-dependent gating of the nv TRPM2 channel; instead it exerts an independent catalytic function which possibly controls the intracellular availability of ADP-ribose.