Institute of Physiology, Medical Faculty, RWTH Aachen, D52057, Aachen, Germany.
Sci Rep. 2017 Aug 3;7(1):7245. doi: 10.1038/s41598-017-07652-4.
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 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 to the pore is decisive which again depends on the open state of the channel. We conclude that separate regulatory processes by Ca on the pore can be discriminated with the aid of 2-APB.
海葵 Nematostella vectensis 的典型 TRPM2 样通道受 ADPR 门控，与其人类同源物相似，但此外还表现出其他脊椎动物 TRPM 通道的特性。因此，它可以帮助我们理解整个亚家族的门控和调节。为了进一步阐明 Ca 作为 ADPR 辅助因子的作用，我们利用了 2-氨基乙基二苯基硼酸盐（2-APB），先前的研究表明，2-APB 以浓度依赖的方式对不同的 TRPM 通道具有抑制或刺激作用，或同时具有这两种作用。2-APB 在高浓度（1mM）下诱导通过 nvTRPM2 的大、非失活电流。在较低浓度（≤0.5mM）下，它阻止了 nvTRPM2 被 ADPR 刺激时的快速电流失活。与它们的起始有相当长的滞后时间相比，这两种作用在 2-APB 洗脱后迅速逆转。对具有修饰的选择性过滤器或降低的 ADP-核糖敏感性的 nvTRPM2 突变体的详细分析表明，2-APB 的作用取决于其进入孔的通道，而通道开放增强了其进入孔的通道。此外，Ca 进入孔的通道是决定性的，这再次取决于通道的开放状态。我们得出结论，Ca 对孔的单独调节过程可以借助 2-APB 来区分。
