Structural insight into TRPV5 channel function and modulation

Abstract

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

Keywords: TRP channel; calcium; calmodulin; cryo-EM.

Fig. 1.

Cryo-EM structure of TRPV5. (A and B) Side (A) and top (B) views of nanodisc-reconstituted TRPV5 1–660 at 2.9-Å resolution, with the subunits colored individually. The nanodisc density surrounds the transmembrane region of the channel (white). (C and D) Side (C) and top (D) views of the TRPV5 tetrameric complex, using the same subunit colors as in A and B. (E) Side view of a TRPV5 monomer with the functional domains colored individually. Structural elements are depicted as ribbons for α-helices, arrows for β-sheets, and ropes for unordered loops. (F) Domain organization of TRPV5, with domains colored as in E. The dashed line denotes a region that was not resolved in the structure but is present in the full-length channel.

Fig. 2.

S1-S2 linker position in TRPV5. (A) Expanded view of the lipid densities observed within the pore domain. Two monomers are shown and individually colored. Lipids are in red. (B) Zoom of the resident lipid in the vanilloid pocket. The density of the lipid is visualized with cyan mesh, and the fitted acyl chain is in cyan. The side chains of residues important in phosphatidylinositol binding in TRPV1 are shown. (C) Zoom-in view of the interaction formed by the S1-S2 linker, annular lipid, and pore domain. The side chains of residues interacting with the lipid are shown. (D) Top view of TRPV5 tetramer showing the positions of the S1-S2 linker (light teal) and the S5-P-S6 domain (pale green). (E and F) Zoom-in view of the intersubunit interface formed by the S1-S2 linker and the pore helix. Putative hydrogen bonds and electrostatic interactions are shown as dashed lines. Side chains of interacting residues are shown as sticks for both, and interatomic distances (F) between side chains are depicted.

Fig. 3.

Pore domain characteristics of TRPV5. (A and B) Top (Left) and bottom (Right) view of TRPV5 full-length (A) and TRPV5 W583A (B). Side chains are shown for two constricting residues in the selectivity filter and lower part of the pore, D542 and I575. (C and D) The ion permeation pathway of closed TRPV5 (C; yellow) and open TRPV5 W583A (D; cyan) is shown as a ribbon diagram with the solvent-accessible space depicted as a pink mesh. Only two subunits are shown for clarity, with side chains of the restricting residues D542 and I575. (E) Diagram of the pore radius calculated with HOLE shown for TRPV5 (yellow) and TRPV5 W583A (cyan). The dotted line indicates the radius of a hydrated calcium ion. (F) Comparison of the TRPV5 (yellow) and TRPV5 W583A (cyan) pore domains with the TRP helix attached. Key residues in the pore domain are depicted.

Fig. 4.

Structure of the TRPV5-CaM complex. (A) Bottom view of TRPV5-CaM with the two subunits including the CaM-interacting one (light teal) and the other two (pale green). CaM is in magenta. (B) Side view of a TRPV5 monomer (light teal) with CaM (magenta) interaction at the C terminus. (C) Overview of CaM (magenta) interacting with two C-terminal helices of TRPV5 (light teal). (D–H) Close-up views of the CaM N-lobe (D and E) and CaM C-lobe (F and G) interacting with the TRPV5 N and C termini. Side chains of hydrophobic interactions are shown as sticks for both CaM (orange) and TRPV5 (blue). In D, interatomic distances between side chains are depicted. (H) CaM-binding assay of HEK293 cells transfected with WT TRPV5 and the indicated mutants. Samples were analyzed by immunoblotting with GFP antibody. The CaM fraction represents the TRPV5 bound to the CaM agarose beads (Top), and input demonstrates TRPV5 expression in total cell lysates (Bottom). A representative immunoblot of three independent experiments is shown. (I) Quantification of the immunoblots is depicted as percentage of WT, which represents the relative CaM binding compared with input.

Fig. 5.

Inactivation of TRPV5 by CaM in a calcium-dependent manner. (A) Cryo-EM density map showing two CaM molecules bound to one TRPV5 channel. (B) Under a basal intracellular calcium level, the C-lobe of CaM with calcium bound is able to interact with the C-terminal distal helix of TRPV5, while the N-lobe is in a calcium-free state. (C) Influx of calcium from TRPV5 increases the intracellular calcium concentration, which allows calcium binding to the N-lobe of CaM and further interaction with the C-terminal proximal helix of TRPV5. This interaction positions the C-lobe of CaM to block the lower gate of the channel. Binding of CaMs to TRPV5 presents a flexible stoichiometry.