Abstract
There are at least two different principles of how ADP-ribose (ADPR) induces activation of TRPM2 channels. In human TRPM2, gating requires the C-terminal NUDT9H domain as ADPR-binding module, whereas in sea anemone, NUDT9H is dispensable and binding of ADPR occurs N-terminally. Zebrafish TRPM2 needs both, the N-terminal ADPR-binding pocket and NUDT9H. Our aim was to pinpoint the relative functional contributions of NUDT9H and the N-terminal ADPR-binding pocket in zebrafish TRPM2, to identify fundamental mechanisms of ADPR-directed gating. We show that the NUDT9H domains of human and zebrafish TRPM2 are interchangeable since chimeras generate ADPR-sensitive channels. A point mutation at a highly conserved position within NUDT9H induces loss-of-function in both vertebrate channels. The substrate specificity of zebrafish TRPM2 corresponds to that of sea anemone TRPM2, indicating gating by the proposed N-terminal ADPR-binding pocket. However, a point mutation in this region abolishes ADPR activation also in human TRPM2. These findings provide functional evidence for an uniform N-terminal ADPR-binding pocket in TRPM2 of zebrafish and sea anemone with modified function in human TRPM2. The structural importance of NUDT9H in vertebrate TRPM2 can be associated with a single amino acid residue which is not directly involved in the binding of ADPR.
Highlights
The Melastatin Type 2 cation channel (TRPM2) represents a unique member of the family of Transient Receptor Potential (TRP) cation channels
It is generally accepted that in human TRPM2, ADPR binds to the NUDT9H domain of the channel[17,18], thereby inducing conformational changes that results in the opening of the pore[6]
During this interaction there is no enzymatic cleavage of ADPR, because the NUDT9H domain of human TRPM2 (hTRPM2) lacks significant catalytic activity[15,16] and channel activation is induced with the non-cleavable ADPR-analogue Alpha-Beta Methylene ADPR (AMPCPR)[16]
Summary
The Melastatin Type 2 cation channel (TRPM2) represents a unique member of the family of Transient Receptor Potential (TRP) cation channels. The ADPR-dependent activation of TRPM2, does not take place via ADP-ribosylation but rather through a different type of interaction in which the C-terminal NUDT9H domain represents a key player, at least in some species variants including the human orthologue[5,6,8,13,14,15,16]. The functional analysis of a far-distantly related species variant from the sea anemone Nematostella vectensis (nvTRPM2) revealed that ADPR-dependent channel gating is possible www.nature.com/scientificreports in the complete absence of the endogenous NUDT9H domain[15,19] These findings directly imply the presence of an additional ADPR interaction site located in the ion channel domain of nvTRPM27,15. In this species variant, the presence of the NUDT9H domain is still indispensable for channel function, its binding affinity to ADPR is greatly reduced[6,8]
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