Molecular basis of multimodal regulation of a TRP channel
Results and Discussion
We identified a TRP channel homolog from a fungus Gibberella zeae, one of the important model organisms for biological studies. This protein, namely TRPGz, was found to response to various cytoplasmic and extracellular stimuli, such as cytosolic Ca2+, membrane potential, extracellular osomotic upchock, oxidizer application, and temperature change. Importantly, these stimuli are the ones commonly activate other eukaryotic TRP channels. Therefore, we expected TRPGz to serve as a prototypic TRP channel, and addressed its molecular bases for multimodal regulation. We identified multiple regulatory modules of TRPGz within its C-terminal cytosolic domain (CTD) that mediate at least part of the multimodal channel functions. One is the middle presumed coiled-coil region necessary for activation by hyperosmolarity and temperature increase, but not for the tetrameric channel formation or other activation modalities. Another is the C-terminal phosphatidylinositol phosphates (PIPs) binding module for channel inhibition. Because the PIPs level in cell membrane dynamically increase upon various cellular stresses, the interactions of PIPs with the PIPs-specific region are likely to play important roles for channel regulation under cellular stress conditions. The NMR analysis of the CTD revealed that most of the CTD is considered to be intrinsically disordered. Nevertheless, we successfully solved the X-ray crystal structure of the presumed coiled-coil region at 1.25 Å resolution using SPring-8 beamlines. The region formed a tetrameric assembly, but the structure is not actually a tetrameric coiled-coil but a four-helix bundle with weaker interprotomer interactions. The analytical ultracentrifugation experiments as well as the NMR analyses confirmed that the CTD exists in association and dissociation equilibrium in the physiological state. Notably, the CTD oligomerization mediated through this bundling region positively correlates with the levels of hyperosmotic- responses. The structural characteristics of the TRPGz-CTD described above, with its intrinsically disordered region and the assembly region with moderate association affinity in the middle, allows dynamic association and dissociation with various binding partners, and thus is considered to be favorable for the multimodal activation commonly observed in TRP channels.
This work proposed structural and biophysical principles for multimodality of a TRP channel family member. The results from this study might contribute to deepen the understanding of the functions TRP channels, which also includes important targets for drug discoveries.
Ihara M, Hamamoto S, Miyanoiri Y, Takeda M, Kainosho M, Yabe I, Uozumi N, Yamashita A. Molecular Bases of Multimodal Regulation of a Fungal Transient Receptor Potential (TRP) Channel. J Biol Chem. 288,15303-17, 2013.