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. 2019 Jan;26(1):40-49.
doi: 10.1038/s41594-018-0168-8. Epub 2018 Dec 31.

Structures of TRPV2 in distinct conformations provide insight into role of the pore turret

Timothy L Dosey  1 , Zhao Wang  1 , Guizhen Fan  2 , Zhixian Zhang  1 , Irina I Serysheva  2 , Wah Chiu  3   4 , Theodore G Wensel  5
Affiliations

Structures of TRPV2 in distinct conformations provide insight into role of the pore turret

Timothy L Dosey et al. Nat Struct Mol Biol. 2019 Jan.

Abstract

Cation channels of the transient receptor potential (TRP) family serve important physiological roles by opening in response to diverse intra- and extracellular stimuli that regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as 'pore turrets', which are proximal to the upper gates. We established the importance of the pore turret through activity assays and by solving structures of rat TRPV2, both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å, respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore-turret-facilitated mechanism.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Fig. 1|
Fig. 1|
Effects of pore turret deletions and phosphoinositides on channel activity. (A) Sequence alignment of the pore turret domains of the TRPV family. Red sequences indicate the deleted regions in the constructs referred to in (B), (C), (D), and (E). Asterisks indicate the first and last residues of the rat TRPV2 pore turret. (B)(D)(E) Dose responses of TRPVs comparing the activities of full-length and pore-turret-deletion variants. Data points are represented as mean ± SEM (n = 3 technical repeats). (C) Dose response for full-length TRPV2 with four amino acid substitutions (F472S, L503F, L510T, Q530E) which confer sensitivity to resiniferatoxin (RTX) and for a pore turret deletion variant with the same substitutions. Data points are represented as mean ± SEM (n = 3 technical repeats). (F) Normalized fluorescent traces for a cell-based Ca2+-flux activity assay demonstrating the potentiation effect of activating a PIP2 phosphatase on TRPV2 activity. Data points are represented as mean ± SEM (n = 3 technical repeats). Empty vector control responses in the precence of low PIP2 levels is indicated by the blackline and the grey line indicates the response with PIP2 at basal levels. (G) Normalized fluorescent traces for a liposome-based Ca2+-flux assay demonstrating the activity of reconstituted full-length TRPV2 and the inhibitory effect of PIP2. Data points are represented as mean ± SEM (n = 3, technical repeats). Empty liposomes responses in the presence of PIP2 is indicated by the grey line and in the absence of PIP2 is indicated by the black line.
Figure 2|
Figure 2|
TRPV2 Constructs and cryo-EM Density Maps (A) (B) WT (blue) and mutant (purple) constructs of TRPV2 used for biochemical characterization and structural determination. Both contain the rat TRPV2 sequence spanning residues 73 to 726; however, the mutant construct has a deletion in the pore turret loop from residues 564 to 589. The coverage of the cryo-EM density maps are in between the residue numbers with an asterisk. (C) Cryo-EM density map for full-length TRPV2 tetramer (blue) with the pore turret (green) visible. (D) Density map for mutant TRPV2 tetramer (purple) showing the missing density in the pore turret region. (E)(F) Models for the WT and mutant TRPV2 monomers fit to segmented maps, and a zoom view showing S1, S2, and S3 helices to display sidechain features.
Fig. 3|
Fig. 3|
Structure and Orientation of Pore Turret Domain (A) The arrangment of the pore turret (green) with respect to the upper gating domains and transmembrane domains of its own subunit (blue) and the transmembrane domains of the adjacent subunit (orange). (B) Side view of the pore turret domain showing its planar structure and perpendicular orientation to the transmembrane domains.
Fig. 4|
Fig. 4|
Cation Pathway of WT and Mutant Variants of TRPV2. (A)(D) S5 and S6 transmembrane segments of the two TRPV2 variants and dot surfaces of the conduction pathways. Black lines indicate the upper and lower gating regions. (B)(E) Van der Vaals radii along the conduction pathway with positions of important residues indicated with arrows and labels. (C)(F) Space filling representation of the WT and Mutant pores looking down through the extracellular side of the channel. Blue, nitrogen; red, oxygen; green, carbons in pore turret; cyan, carbon in rest of WT model; magenta, carbon in mutant model.
Fig. 5|
Fig. 5|
Comparisons of TRPV2 structures. Cyan, WT; grey, (A, B, E), fully-closed (PDB: 5AN8); magenta, (C, D, F), Mutant. (A, C) full model alignments based on S1 and S2 positions. (B, D) Voltage-sensing-like domain (VSLD) region showing a shift in the S3 position between the open and closed structures. (E, F) Alignment of Pipes and planksrepresentations of the ankyrin repeat domains.
Fig. 6|
Fig. 6|
Model alignments of S3-S6. Alignments are based on S1 and S2 as in Fig. 5.(A)(E) Alignments of S3-S6 of fully closed TRPV2 sructure (grey) (5AN8) and partially open mutant TRPV2 (purple) with the fully-open WT TRPV2 structure (blue) (B)(F) Alignments of the upper gate including the pore helix, selectivity filter, pore loop, and upper S6 regions. (C) (G) View of the aligned S4/S5 linker domains and their relative positions to S6 and the TRP domain. (D) (H) Relative positions of the S5 and pore helices.
Figure 7|
Figure 7|
Model Mechanism for agonist-independent TRPV2 activation. (A) Top view of the TRPV2 tetramer looking down the cation pathway from the extracellular side. (B) Sideview for a crossection of TRPV2 displaying two apposing subunits.
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