A First Look into the 3D Structure of the TRPV2 Channel by Single-Particle Cryo-Em

Abstract

The TRPV2 channel is a ∼385 kDa integral membrane protein complex that functions as a high-threshold thermosensor and is involved in calcium signaling during inflammation and nerve injury. TRPV2 is constituted of four subunits, each with six transmembrane (TM) spanning helices with a pore loop between the fifth and sixth TM helices. To date, little is understood about the complex biophysical properties or the 3D architecture of the TRPV2 channel. We used single particle electron cryo-microscopy (cryo-EM) to solve the 3D structure of the tetrameric TRPV2 channel. To obtain sufficient quantities of purified channel proteins for structural and functional analysis, we used heterologous expression in the budding yeast, S. cerevisiae and immunoaffinity chromatography to purify TRPV2. The purified channel was vitrified in the presence of amphipathic polymer, amphipol 8-35, which was used to substitute for solubilizing detergent in TRPV2 preparations. This procedure allowed us to achieve optimal ice thickness and uniform particle distribution in the cryospecimen. Vitrified TRPV2 samples were imaged using an FEI Polara electron cryo-microscope operated at 300 keV at liquid-nitrogen temperature. Images of the channel were acquired on a 4k x 4k CCD camera (TVIPS) at 59,000 nominal magnification of microscope using low-dose mode and a defocus range of 2-5 μm. The 3D reconstruction of TRPV2 was generated with 20,608 particle images extracted from 431 CCD frames, and image processing was performed using EMAN2. The resulting structure was validated using tilt-pair analysis, and is interpretable to ∼15 A based on gold standard resolution criterion. Supported by grants from NIH (R21AR063255, R01GM072804, P41GM103832, T32KD007696, R01EY07981) and by AHA (12GRNT10510002).