Abstract
The archetypal TRPM2-like channel of the sea anemone Nematostella vectensis is gated by ADPR like its human orthologue but additionally exhibits properties of other vertebrate TRPM channels. Thus it can help towards an understanding of gating and regulation of the whole subfamily. To elucidate further the role of Ca2+ as a co-factor of ADPR, we exploited 2-aminoethyl diphenylborinate (2-APB), previously shown to exert either inhibitory or stimulatory effects on diverse TRPM channels, or both in a concentration-dependent manner. 2-APB in high concentrations (1 mM) induced large, non-inactivating currents through nvTRPM2. In lower concentrations (≤0.5 mM), it prevented the fast current inactivation typical for nvTRPM2 stimulated with ADPR. Both these effects were rapidly reversed after wash-out of 2-APB, in contrast to a considerable lag time of their onset. A detailed analysis of nvTRPM2 mutants with modified selectivity filter or reduced ADP-ribose sensitivity revealed that the actions of 2-APB depend on its access to the pore which is enhanced by channel opening. Moreover, access of Ca2+ to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca2+ on the pore can be discriminated with the aid of 2-APB.
Highlights
During evolution, the progenitors of cnidarians and of vertebrates have separated an estimated time of 800 million years ago[5]
We have provided evidence that in nvTRPM2, the NUDT9H domain regulates the intracellular availability of ADPR by its enzymatic activity, whereas a further, yet un-identified interaction site for ADPR is present elsewhere in the channel protein[6]
To get further insights into the gating mechanism of the archetypal TRPM channel from Nematostella vectensis, we tested the effect of 2-aminoethyl diphenylborinate (2-APB), a non-specific TRP channel modulator, which has widely been used as a pharmacological tool to explore intracellular Ca2+ signaling16–18. 2-APB may exert either activating or inhibitory functions on closely related TRPM channels (e.g. TRPM6 vs. TRPM719, or human TRPM7 vs. zebrafish TRPM720)
Summary
The progenitors of cnidarians and of vertebrates have separated an estimated time of 800 million years ago[5] In spite of this long time for a divergent development, nvTRPM2 and hTRPM2 are both activated by ADPR, which is not known as a stimulus for any other ion channel[4]. The molecular basis that underlies the discrepant effects of 2-APB on TRPM channels has not been investigated We hypothesized that these are related to a specific triplet of amino acid residues within the proximal pore loop that exists in two principal versions within the TRPM family[3]. Since activation as well as inactivation of nvTRPM2 is Ca2+-dependent, the present study establishes 2-APB as a valuable tool by which diverse modulatory processes by Ca2+ within the channel pore can be disentangled. It is hoped that these and further ongoing studies will help towards an understanding of some aspects of TRPM channel regulation in an integrative sense which applies to the whole family, under consideration of specific common and divergent structural and functional properties of individual members
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