3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method

Abstract

The TRPV1 is a nonselective cation channel that responds to various signals, including high temperature with threshold at 43 °C, acidic conditions, as well as chemical compounds such as capsaicin and allylisothiocyanate. Because TRPV1 is a membrane protein with large molecular mass, crystallography technique has not been succeeded for a long time. However recent progress in the cryoelectron microscopy enabled depiction of the TRPV1 structure in atomic resolution. Cryo-EM and the single particle reconstruction technique showed structures of apo- and ligand binding forms of TRPV1 with various agonists, antagonists and toxins. In spite of accumulation of structural data, gating mechanisms of TRPV1 is not clearly understood yet. Information about state-to-state transition is missing. To understand the dynamics of TRPV1 in channel function, we adopted the Diffracted X-ray Tracking (DXT) technique to this protein. In DXT, individual protein was labeled with gold nanocrystals, and the motion of X-ray diffraction spots from the crystal were investigated as intramolecular movement of TRPV1 in real time. We introduced “Met tag” for labeling nanocrystal and “His tag” for substrate absorption to TRPV1. It was expressed in HEK293 cells, purified, and immobilized on the Ni-NTA coated polyimide substrates. Intramolecular motion was recorded by tracking the movement of diffraction spots from the gold nanocrystals. Molecular dynamics of TRPV1 against capsaicin, pH change, and temperature activation were investigated.