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. 2019 Jul 24;5(7):eaaw7935.
doi: 10.1126/sciadv.aaw7935. eCollection 2019 Jul.

Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

Jingjing Duan  1   2   3 , Jian Li  4   5 , Gui-Lan Chen  6   7 , Yan Ge  6 , Jieyu Liu  6 , Kechen Xie  6 , Xiaogang Peng  8 , Wei Zhou  8 , Jianing Zhong  5 , Yixing Zhang  2 , Jie Xu  9 , Changhu Xue  9 , Bo Liang  10 , Lan Zhu  11 , Wei Liu  11 , Cheng Zhang  12 , Xiao-Li Tian  1 , Jianbin Wang  2 , David E Clapham  3 , Bo Zeng  6   7 , Zongli Li  13 , Jin Zhang  2
Affiliations

Cryo-EM structure of TRPC5 at 2.8-Å resolution reveals unique and conserved structural elements essential for channel function

Jingjing Duan et al. Sci Adv. .

Abstract

The transient receptor potential canonical subfamily member 5 (TRPC5), one of seven mammalian TRPC members, is a nonselective calcium-permeant cation channel. TRPC5 is of considerable interest as a drug target in the treatment of progressive kidney disease, depression, and anxiety. Here, we present the 2.8-Å resolution cryo-electron microscopy (cryo-EM) structure of the mouse TRPC5 (mTRPC5) homotetramer. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. The disulfide bond at the extracellular side of the pore and a preceding small loop are essential elements for its proper function. This high-resolution structure of mTRPC5, combined with electrophysiology and mutagenesis, provides insight into the lipid modulation and gating mechanisms of the TRPC family of ion channels.

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Figures

Fig. 1
Fig. 1. Overall structure of mouse TRPC5.
(A) Cryo-EM density map of mTRPC5 at 2.8 Å overall resolution with each monomer represented in different colors (left, side view; right, top view). (B) Ribbon diagrams of the mouse TRPC5 model with the channel dimensions indicated. (C) Ribbon diagrams depicting structural details of a single subunit. (D) Linear diagram depicting the major structural domains of the TRPC5 monomer, color-coded to match the ribbon diagram in (C). ARD, ankkyrin repeat domain.
Fig. 2
Fig. 2. TRPC5 ion conduction pathway compared with TRPC4 and other TRPCs.
(A) Side view of TRPC5’s pore region with chains A and C compared with TRPC4 (gray). The ion conduction pathway is shown as dots and mapped using HOLE with key amino acid residues labeled. (B) Pore radius along the central axis. The side chains of glycine form a narrow constriction at the selectivity filter. “INQ” motif forms the lower gate. (C) Side view of TRPC5 (blue) monomer subunit compared with TRPC4 (gray). Differences in the organizations of a linker (red arrowheads) of S5 and the small loop above the pore helix of the extracellular pore domain are enlarged. (D) Sequence of the mouse TRPC5 aligned to TRPC4 and other TRPC subfamily members (Clustal Omega) between S5 and S6 including the linker, pore helix, and pore loop. Regions corresponding to different extracellular pore domains are indicated by the red arrowheads. The two cysteines forming disulfide bonds, a conserved “LFW” motif, and the selectivity filter are highlighted. (E to G) Patch clamp recordings of TRPC5 mutants in response to channel activators Gd3+ and EA.
Fig. 3
Fig. 3. Comparison of the TRPC5 structure with other TRP channel structures.
(A) Superimposed side views of mouse TRPC5 subunit (blue) compared with other TRPC family members, including mouse TRPC4 [Protein Data Bank (PDB) ID code 5Z96, gray] (19), human TRPC3 (PDB ID 5ZBG, pink) (20), and human TRPC6 (PDB ID 5YX9, yellow) (21). (B) The conserved LFW motif in the pore helix from a π-π interaction that stabilizes the pore region. (C) Key pore loop disulfide bond between Cys553 and Cys548 in TRPC5 (black arrowhead) and the corresponding pore loop disulfide bond in TRPC4 that close to a linker (red arrowheads) of S5 and loop above the pore helix. This disulfide bond is not present in TRPC3 or TRPC6. (D) Differences in the organization of the S3 helix between the TRPC4/5 and TRPC3/6. The S3 helices of TRPC3 and TRPC6 are longer than those of TRPC5 and TRPC4. (E) Patch clamp recordings of wild-type (WT) TRPC5 and cysteine mutants in response to the reducing agent [dithiothreitol (DTT)] and channel activator (EA). (F) Localization of enhanced yellow fluorescent protein–tagged WT TRPC5 and cysteine mutants in HEK293 cells. (G) Quantification of cell surface expression of TRPC5 cysteine mutants (n = 3). Mean values are shown as gray bars. By analysis of variance (ANOVA), there is no statistically significant difference. a.u., arbitrary units.
Fig. 4
Fig. 4. Mutations in the loop preceding the disulfide bond of TRPC5 alter channel inactivation kinetics.
(A to F and I) The time course of whole-cell currents measured at +80 and −80 mV and representative I-V relationships in different conditions as labeled. TRPC5-C4motif, TRPC5 mutant carrying the TRPC4 motif ETKGLS; TRPC4-C5motif, TRPC4 mutant with the TRPC5 motif TRAIDEPNN; TRPC5-C4motif-C553A-C558A, TRPC5 mutant bearing the TRPC4 motif and double-cysteine mutations. (G and H) Time constants (τ) of channel activation and inactivation by 100 nM EA. τ values from each recording are plotted as dots. “∞” indicates that the current did not decay in the recording time frame. Mean τ values of each mutant are shown as gray bars. There is no significant difference by ANOVA in (G) (n = 8 to 11).
Fig. 5
Fig. 5. The cation binding site in TRPC5.
(A) A cation (purple sphere) on the cytosolic face is in the hydrophilic pocket of the S1-S4 domain, interacting with Glu418, Glu421, Asn436, and Asp439. Enlarged view of the cation binding site. (B) Comparable Ca2+ (green sphere) binding site and an enlarged view of TRPM4 as “E/Q/N/D” (TRPC5, “E/E/N/D”). (C) Sequence of the mouse TRPC5 aligned to mTRPC4 and other representative TRP members (Clustal Omega). The key residues are highlighted as E/E/N/D, which are conserved in TRPC2/3/5/6/7 and TRPM7 but as E/Q/N/D in TRPC4 and TRPM4. (D) Patch clamp recordings of TRPC5 mutants in response to the channel activators Gd3+ and EA. For the N436R mutant, 12 transfected cells showed no response to EA; weak activation was observed in three cells.
Fig. 6
Fig. 6. Lipid coordination in TRPC5.
Lipid-channel interactions. (A) Side and top views of ribbon diagrams of the TRPC5 tetramer: four CHS molecules and four PLs (potentially ceramide-1-phosphate or phosphatidic acid) are shown as spheres with purple or yellow carbons, respectively. (B) Side views of each CHS and PL molecules per protomer. (C and D) Ribbon diagram of the TRPC5 lipid binding regions. (C) PL interacts with the pore helix through Trp577 and Phe576. (D) CHS, shown in purple, interacts with the S4/S5 linker at Asn500 and the N-terminal domains at Trp315, Tyr316, and Trp322. (E and F) Patch clamp recordings of lipid binding site mutants in response to EA.
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