Modulation of activation and inactivation by Ca(2+) and 2-APB in the pore of an archetypal TRPM channel from Nematostella vectensis

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 Ca(2+) 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 Ca(2+) to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca(2+) on the pore can be discriminated with the aid of 2-APB.