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. 2008 May 27;105(21):7451-5.
doi: 10.1073/pnas.0711835105. Epub 2008 May 19.

Structure of TRPV1 channel revealed by electron cryomicroscopy

Vera Y Moiseenkova-Bell  1 , Lia A Stanciu, Irina I Serysheva, Ben J Tobe, Theodore G Wensel
Affiliations

Structure of TRPV1 channel revealed by electron cryomicroscopy

Vera Y Moiseenkova-Bell et al. Proc Natl Acad Sci U S A. .

Abstract

The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-A structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Purification of TRPV1 from S. cerevisiae membranes. (a) SDS/PAGE analysis of TRPV1 eluted from an immunoaffinity column (lane 1) and Western blotting of the elution fraction with 1D4 antibody (lane 2). The minor lower band contains the C-terminal 1D4 epitope and may result from proteolysis at the N terminus. (b) Representative gel-filtration trace of TRPV1 on Superdex 200 HiLoad 16/60 column. The migration positions of molecular mass standards (dashed line) and a least-squares fit of elution volume (solid line) vs. molecular mass are shown. Amounts of TRPV1 in each fraction were determined by Western blot analysis with 1D4 antibody. The calculated molecular mass of TRPV1 including bound detergent is ≈500 kDa, in good agreement with the position of the major peak. Sizes of molecular mass standards are shown on the plot (650, 158, and 44 kDa).
Fig. 2.
Fig. 2.
Agonist-induced Ca2+ efflux from TRPV1 vesicles. (a) Fura-2 excitation spectra measured before (dashed line) and immediately after (solid line) application of 50 nM resiniferotoxin (RTX) to the cuvette containing TRPV1 vesicles loaded with 5 mM internal Ca2+. Ionomycin (2 μM) induced changes represented by the small-dashed line. (b) The same experiment with protein-free vesicles prepared identically but without TRPV1 reveals no release of Ca2+ by RTX (n = 6). (c) Image of TRPV1 vesicles.
Fig. 3.
Fig. 3.
Single-particle electron microscopy of TRPV1. (a) Typical field from a raw image of a frozen solution of the channel, with individual protein images, or particles, surrounded by white rectangles. (b) Comparison of class-averaged images (second and fourth columns) with projections of the final 3D model (first and third columns). (c) Three-dimensional map of TRPV1 in an isosurface representation with the contour level chosen to enclose a molecular volume corresponding to a mass of 500 kDa.
Fig. 4.
Fig. 4.
Distribution of particle orientations over the asymmetric unit used in the reconstruction. Brighter dots denote a larger number of particles (minimum 35; maximum 200).
Fig. 5.
Fig. 5.
The proposed location of the 3D structure of TRPV1 in the plasma membrane. A vertical cut-away view shows the internal mass distribution of the protein.
Fig. 6.
Fig. 6.
Comparison of 3D reconstruction of TRPV1 to atomic structures of Kv1.2 potassium channel and TRPV1 ankyrin repeats. (a) Docking of the high-resolution structure of Kv1.2 transmembrane domains (maroon; PDB entry 2A79) and TRPV1 ankyrin domains (green; PDB entry 2PNN; two of four such domains are shown) into TRPV1 3D reconstruction. (b) Representation of x-ray structure of Kv1.2 (PDB entry 2A79) at 19-Å resolution (maroon, transmembrane domains; blue, T1 domain and β-subunit).
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References

    1. Cosens DJ, Manning A. Abnormal electroretinogram from a Drosophila mutant. Nature. 1969;224:285–287. - PubMed
    1. Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus: A putative integral membrane protein required for phototransduction. Neuron. 1989;2:1313–1323. - PubMed
    1. Montell C. The TRP superfamily of cation channels. Sci STKE. 2005;272:re3. - PubMed
    1. Nilius B, Voets T. TRP channels: A TR(I)P through a world of multifunctional cation channels. Pflügers Arch. 2005;451:1–10. - PubMed
    1. Voets T, Talavera K, Owsianik G, Nilius B. Sensing with TRP channels. Nat Chem Biol. 2005;1:85–92. - PubMed

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