Abstract
The human redox-sensitive Transient receptor potential melastatin type 2 (hTRPM2) 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 hTRPM2. 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, Ca2+-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 nvTRPM2 to ADP-ribose. The deletion, however, created channels that were activated by hydrogen peroxide, as did mutations within the NUDT9H domain of nvTRPM2 that presumably suppress its enzymatic function. The same findings were obtained with the nvTRPM2 channel when the NUDT9H domain was replaced by the corresponding sequences of the original hNUDT9 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 nvTRPM2 in co-expression experiments with the C-terminally truncated nvTRPM2 channel. Thus, there is a clear correlation between the loss of enzymatic activity and the capability of nvTRPM2 to respond to oxidative stress. In striking contrast, the channel function of the hTRPM2 orthologue, in particular its sensitivity to ADP-ribose, was abrogated by already small changes of the NUDT9H domain. These findings establish nvTRPM2 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 nvTRPM2 channel; instead it exerts an independent catalytic function which possibly controls the intracellular availability of ADP-ribose.
Highlights
The human Transient receptor potential melastatin-related 2 channel is a Ca2+ permeable cation channel which has been described as celluar redox sensor [1]
The results demonstrate that the NUDT9 domain of nvTRPM2 is not directly involved in adenosine 5’-diphosphoribose (ADPR)-dependent channel gating, whereas it has a crucial role in preventing responses to H2O2 which is regarded to induce intracellular accumulation of ADPR [16]
In strict contrast to the situation in human Transient receptor potential melastatin-related 2 (hTRPM2) where NUDT9 homology (NUDT9H) is decisive for channel activation by ADPR and by H2O2, its role in nvTRPM2 seems to be independent from channel gating and confined to the control of intracellular ADPR concentrations
Summary
The human Transient receptor potential melastatin-related 2 (hTRPM2) channel is a Ca2+ permeable cation channel which has been described as celluar redox sensor [1]. The TRPM orthologue of N. vectensis shares distinct similarities with the S1-S2 transmembrane linker of the human TRPM3 channel [5], it should be indicated as TRPM2 because its C-terminus contains the characteristic and functional decisive structural element of the hTRPM2 channel, the NUDT9 homology (NUDT9H) domain [9;10]. This domain, with striking homology to the human adenosine 5’-diphosphoribose (ADPR) pyrophosphatase NUDT9 of the Nudix hydrolase family [11] controls the activation of hTRPM2 by intracellular ADPR [9;12]. H2O2 which is an established further activator of hTRPM2 [1,13], was completely without effect on nvTRPM2 [5]
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