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. 2014 Feb 4;22(2):260-8.
doi: 10.1016/j.str.2013.11.008. Epub 2013 Dec 26.

Structural insight into the assembly of TRPV channels

Kevin W Huynh  1 , Matthew R Cohen  2 , Sudha Chakrapani  2 , Heather A Holdaway  1 , Phoebe L Stewart  1 , Vera Y Moiseenkova-Bell  3
Affiliations

Structural insight into the assembly of TRPV channels

Kevin W Huynh et al. Structure. .

Abstract

Transient receptor potential (TRP) proteins are a large family of polymodal nonselective cation channels. The TRP vanilloid (TRPV) subfamily consists of six homologous members with diverse functions. TRPV1-TRPV4 are nonselective cation channels proposed to play a role in nociception, while TRPV5 and TRPV6 are involved in epithelial Ca²⁺ homeostasis. Here we present the cryo-electron microscopy (cryo-EM) structure of functional, full-length TRPV2 at 13.6 Å resolution. The map reveals that the TRPV2 cytoplasmic domain displays a 4-fold petal-like shape in which high-resolution N-terminal ankyrin repeat domain (ARD) structures can be unambiguously fitted. Fitting of the available ARD structures for other TRPV subfamily members into the TRPV2 EM map suggests that TRPV subfamily members have highly homologous structural topologies. These results allowed us to postulate a structural explanation for the functional diversity among TRPV channels and their differential regulation by proteins and ligands.

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Figures

Figure 1
Figure 1. Single-Channel Properties of Liposome-Reconstituted TRPV2
(A) Excised inside-out patches of proteoliposomes containing TRPV2 channels. The channels were activated by 100 μM probenecid (PRB) in the pipette. Single-channel currents were recorded under symmetrical KCl concentrations (150 mM) and the membrane potential was held at +50 mV. Channel activity was blocked by 100 μM GdCl3 applied using a rapid solution exchanger. (B) Representative single-channel traces measured at indicated membrane potentials. Current-voltage relationship showed a single-channel conductance of 304 ± 4 pS (n = 4).
Figure 2
Figure 2. Cryo-EM Imaging and Reconstruction of TRPV2
(A) Cryo-electron micrograph of monodisperse TRPV2 particles. White boxes represent typical views of TRPV2. Scale bar, 100 nm. (B) Representative reference-free class averages of TRPV2 generated in IMAGIC (van Heel et al., 1996). (C) Reprojections of the initial 3D model without imposed symmetry corresponding to the reference-free class averages. (D) Side views, top view, and bottom view of the cryo-EM reconstruction of TRPV2 at 13.6 Å resolution. The structure shows a well-defined cytoplasmic region that displays a petal-like arrangement. The dashed lines represent the plasma membrane. Scale bar, 25 Å.
Figure 3
Figure 3. N-Terminal ARD Arrangement in the TRPV2 Map
(A and B) Side view (A) and bottom view (B) of the TRPV2 EM map with ARD crystal structures (Jin et al., 2006; PDB ID code: 2ETB) docked by Colores into the cytoplasmic densities. Each color represents an individual subunit. (C and D) Side view (C) and bottom view (D) of the calculated ARD electrostatic potential (Baker et al., 2001) docked into the TRPV2 map. Positive potentials are shown in blue, negative potentials in red. Scale bar, 25 Å.
Figure 4
Figure 4. Fitting of Homologous TM Domain Structure and Prediction of the Location of the MNG Detergent Belt, N-Terminal Linker Region, and C Terminus in the TRPV2 Map
(A) Crystal structure of the MlotiK1 TM domain (Clayton et al., 2008; PDB ID code: 3BEH) docked into the central TM density of the TRPV2 EM map. The extra density around the central TM region outlined in dark gray (labeled as D) represents the uneven 15–20 Å MNG detergent belt. Dashed line represents the cross-section depicted in (B). (B) A cross-section through the TM domain clearly shows the detergent belt (in mesh labeled as D) relative to the central density where the MlotiK1 TM domain was docked. More cross-sections through the TM region of the density are shown in Figure S5. (C) Side view of the TRPV2 EM map fitted with the MlotiK1 TM domain and TRPV2 ARD crystal structures. Dashed box represents the possible location of the TRPV2 N terminus linker region and C terminus. A cross-section of this segment is depicted in (D) and marked with asterisks. (D) A cross-section through the TRPV2 structure showing the possible location of the TRPV2 N terminus linker region and C terminus. More cross-sections through this portion of the EM density are shown in Figure S5. Scale bar, 25 Å.
Figure 5
Figure 5. Comparison of TRPV ARD Structures Fitted into the TRPV2 EM Map
Surface representation of the MlotiK TM domain (brown; PDB ID code: 3BEH) and (A) TRPV2 ARDs (PDB ID code: 2ETB), (B) TRPV6 ARDs (PDB ID code: 2RFA), (C) TRPV4 ARDs in the absence of ATP (PDB ID code: 4DX1), and (D) TRPV4 ARDs in the presence of ATP (PDB ID code: 4DX2), fitted into the EM map. The concave ligand-binding surface on the ARDs is highlighted in red (TRPV2), purple (TRPV6), yellow (TRPV4 ATP-free), and blue (TRPV4 ATP-bound). The ATP molecule is represented in orange and Ca2+ ions are represented in green.
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