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. 2010 Apr 9;285(15):11210-8.
doi: 10.1074/jbc.M109.090712. Epub 2009 Dec 31.

A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy

Hideki Shigematsu  1 , Takaaki Sokabe, Radostin Danev, Makoto Tominaga, Kuniaki Nagayama
Affiliations

A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy

Hideki Shigematsu et al. J Biol Chem. .

Abstract

The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.

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Figures

FIGURE 1.
FIGURE 1.
Expression profile and [Ca2+]i changes in Sf9 cells infected with baculovirus containing His-rTRPV4. a, Western blot analysis of expression of His-rTRPV4 by using an anti-hexahistidine antibody is shown. Differences in post-infection time and m.o.i. are shown. (i) shows the difference in m.o.i. at 72 h post-infection. (ii) shows the difference in post-infection time at an m.o.i. of 5.0. b, shown is fluorescence imaging of Sf9 cells infected with baculovirus containing His-rTRPV4 observed during heat and agonist stimulation. Pseudocolored images represent the 340/380-nm ratio for Fura-2 fluorescence. The corresponding traces during stimuli are shown below. The period of each stimulus is indicated above the traces, and the measured temperature changes are plotted at the bottom.
FIGURE 2.
FIGURE 2.
Oligomerization analysis of purified His-rTRPV4. a, shown is a size-exclusion chromatogram of proteins in the detergent-solubilized state after affinity purification. The retention volume of the molecular mass standards is shown by crosses for thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). The molecular mass of His-rTRPV4 (including detergent molecules) was estimated as 500 kDa. b, SDS-PAGE analysis of the input, fraction 1, and fraction 2 indicated in the size-exclusion chromatogram was assessed by Coomassie staining. c, chemical cross-linking analysis of purified His-rTRPV4 was assessed by Coomassie staining (left) and a Western blot using anti-His-tag antibody (right). The molecular weight of a cross-linked band above 390 kDa is consistent with a tetramer of His-rTRPV4 subunits (103 kDa). d, shown is a general view of a Nanogold-labeled and NanoVan-stained His-rTRPV4 and an enlarged view of particles having multiple Nanogolds.
FIGURE 3.
FIGURE 3.
Cryo-EM observation of His-rTRPV4 in the detergent-solubilized state. A typical raw image of purified His-rTRPV4 (left) is shown. Some of the individual proteins (white arrows) were picked from the raw image, as shown on the right.
FIGURE 4.
FIGURE 4.
Three-dimensional reconstruction using 4-fold symmetry. a, shown is a surface representation of rTRPV4 with the contour level enclosing a volume corresponding to 500 and 630 kDa for the gray solid surface and gray mesh, respectively. b, Fourier shell correlation (FSC) is shown between reconstructions from even and odd halves of the data set, plotted against spatial frequency. The value falls to the criterion level of 0.5 at a resolution of 3.5 nm. c, shown is Euler angular distribution over the asymmetric unit used in the reconstruction. The brightness of each dot corresponds to the number of particles belonging to the class average Black represents 7 particles, and white represents 43 particles. d, shown is a comparison of class averages (left) with projections of the final three-dimensional model (right). Corresponding Euler angles and numbers of particles were indicated at the right.
FIGURE 5.
FIGURE 5.
The shape and dimensions of surface representation of His-rTRPV4. a, shown is a surface representation of rTRPV4 viewed from four different angles. Dimensions and estimated of volume analysis (italics) are shown. b, shown are surface representations of the transmembrane region of 6TM tetrameric cation channels, TRPV4 (gray), MlotiK1 (blue, PDB code 3BEH), and Kv1.2-chimera (yellow, PDB code 2R9R). Surface representations calculated from atomic coordinates are shown at 3 nm of resolution and superimposed with its ribbon diagram. The upper three models show extracellular views of transmembrane regions, and the lower three show side views. The surface representation of transmembrane region of TRPV4 is superimposed with gray-meshed entire structure.
FIGURE 6.
FIGURE 6.
Superposition of high resolution structures onto the three-dimensional model of TRPV4. Shown is a superposition of a tetramer of the transmembrane segments of MlotiK1 (PDB code 3BEH) onto the small component and a tetramer of the ARD of TRPV1 (PDB code 2PNN) onto the large component of our 3.5-nm map of TRPV4. Superposition onto surface representation is shown as a top view (a), a bottom view (b), and a side view (c). Superposition onto the density map is shown as a side-view projection (d), and horizontal cross-sections are parallel to the membrane plane (e). Each section is 1.3-nm thick. The number in each section corresponds to that on the left side of d. A bird's-eye view of the superposition of MlotiK1 monomer (blue) onto our 3.5-nm map of TRPV4 is shown. 4α-PDD binding pockets are colored with purple and green for TM segments 3 and 4, respectively.
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