ARTICLE DOI: 10.1038/s41467-018-05247-9 OPEN Structure of the mouse TRPC4 ion channel Jingjing Duan1,2, Jian Li1,3, Bo Zeng 4, Gui-Lan Chen4, Xiaogang Peng5, Yixing Zhang1, Jianbin Wang1, David E. Clapham 2, Zongli Li6 & Jin Zhang1 Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intra- cellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. 1234567890():,; The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels. 1 School of Basic Medical Sciences, Nanchang University, 330031 Nanchang, Jiangxi, China. 2 Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA. 3 Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA. 4 Key Laboratory of Medical Electrophysiology, Ministry of Education, and Institute of Cardiovascular Research, Southwest Medical University, 646000 Luzhou, Sichuan, China. 5 The Key Laboratory of Molecular Medicine, The Second Affiliated Hospital of Nanchang University, 330006 Nanchang, China. 6 Department of Biological Chemistry and Molecular Pharmacology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA. These authors contributed equally: Jingjing Duan, Jian Li, Bo Zeng. Correspondence and requests for materials should be addressed to Z.L. (email: [email protected]) or to J.Z. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:3102 | DOI: 10.1038/s41467-018-05247-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05247-9 ammalian transient receptor potential (TRP) channels Table 1 Data collection and refinement statistics Mare activated by a wide spectrum of ligands, tempera- ture, lipids, pH, and as yet unknown stimuli. They fi Cryo-EM of TRPC4 [EMD:6901] are classi ed into six subfamilies based on sequence similarity: [PDB:5Z96] TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), Data collection and processing TRPA (ankyrin), TRPML (mucolipin), and TRPP (or PKD) (polycystin)1. The TRPC subfamily are non-selective cation Microscope FEI Tecnai Polara channels (Na+,K+,Ca2+) that alter proliferation, vascular tone, Detector Gatan K2 fi and synaptic plasticity2,3. This family can be further subdivided Calibrated magni cation 40,607 Voltage (kV) 300 into two subgroups: TRPC2/3/6/7 and TRPC1/4/5. TRPC4 is Electron exposure (e–/Å2) 52.8 broadly expressed in human tissues and can assemble as homo- Defocus range (μm) 1.5–3.0 meric channels or form heteromeric channels with TRPC1 and – Pixel size (Å) 1.23 TRPC54 7. Studies of Trpc4-deficient mice have shown that Symmetry imposed C4 TRPC4 affects endothelial-dependent regulation of vascular tone, Initial particle images (no.) 381,165 endothelial permeability, and neurotransmitter release from tha- Final particle images (no.) 232,858 8 FSC threshold 0.143 lamic interneurons . Stimulation of Gq and Gi/oG-protein- coupled receptors (GPCRs) as well as tyrosine kinase receptors Map resolution (Å) 3.3 fi potentiate channel activity9,10. Activation of TRPC4 is regulated Re nement by intracellular Ca2+, phospholipase C, and membrane lipids by Refinement software phenix.real_space_refine unclear mechanisms. In addition, Storch et al.11 have proposed a Initial model used (PDB code) De novo potential mechanism related to phosphatidylinositol 4,5-bispho- Model composition + + sphate and Na /H exchanger regulatory factor proteins11. Non-hydrogen atoms 21,506 Along with the revolution in cryo-electron microscopy (cryo- Protein residues 2608 EM), improved sample preparation, data acquisition, and image Ligands 12 2 processing strategies, the structures of TRPVs12–17, TRPA118, B factors (Å ) – TRPP119, TRPML120, TRPM421 24, and TRPM825 have been Average 65.46 solved. Here we present the structure of mouse TRPC4 in its apo r.m.s. deviations state at pH 7.5 at an overall resolution of 3.3 Å. The structure Bond lengths (Å) 0.01 provides detailed information on the ion permeation, selectivity, Bond angles (°) 1.11 and gating mechanism of TRPC subfamily. Validation MolProbity score 1.83 Clashscore 7.25 Results Ramachandran plot Overall structure of the mouse TRPC4 tetrameric ion channel. Favored (%) 97.39 The mouse TRPC4 (residues amino acids (a.a.) 1–758, excluding Allowed (%) 2.08 a.a. 759–974) was expressed using the BacMam expression system Disallowed (%) 0.53 (Methods) and purified protein (pH 7.5) was used for single- particle cryo-EM analysis (Supplementary Fig. 1). The overall resolution of TRPC4 reconstruction was 3.3 Å (Supplementary the curve between 10 and 40 mV due to outward Mg2+ block26. Fig. 2 and Table 1), which enabled us to construct a near-atomic These results suggest that, in the context of chemical modulation, model (Supplementary Fig. 3). Disordered regions led to poor the biophysical properties of the truncated construct are similar densities for 4 residues in the S1–S2 loop, 2 residues in the S3–S4 to that of the full-length TRPC4. loop, 27 residues in the distal N terminus, and 28 residues in the To futher test the functional properties of the truncated truncated distal C terminus. In total, the TRPC4 structure is a construct, we examined receptor-operated activation of TRPC4 four-fold symmetric homotetramer (Fig. 1a) with dimensions of by coexpression with GPCRs. In cells transfected with TRPC4 100 Å × 100 Å × 120 Å (Fig. 1b). Each monomer consists of a constructs and P2Y1 and P2Y2 receptors (both coupled to Gq transmembrane domain (TMD) and a compact cytosolic domain. proteins), extracellular application of ADP (P2Y1 agonist) or The cytosolic domain is composed of two subdomains: the N- ATP (P2Y2 agonist) induced large currents for full-length terminal subdomain consisting of four ankyrin repeats TRPC4. In contrast, the currents for truncated TRPC4 were (AR1–AR4) and seven α-helices (H1–H7), and the C-terminal much smaller but still exhibited the characteristic I–V relation- subdomain containing a connecting helix and a coiled-coil ship of the TRPC4 channel (Supplementary Fig. 4a). The domain (Fig. 1c, d). truncated TRPC4 did not respond to Gi/o receptor agonists Ca2+ measurements and electrophysiological studies were DAMGO (μOR) or carbachol (M2R) (Supplementary Fig. 4b). performed to verify that the truncated construct used for structural investigation (residues a.a. 1–758, excluding a.a. 759–974) was permeable to cations and retained sensitivity to Major structural differences with other TRP subfamilies.In channel activator englerin A, blocker ML204, and blocker 2-APB Fig. 3, we compare the TRPC4 structure with previously reported (Fig. 2). Englerin A induced a robust rise of intracellular Ca2+ in TRP structures. Not surprisingly, the organization of six helices in both full-length and truncated TRPC4-transfected 293T cells, but each TMD is similar to that of other TRP channels, while the not in the empty vector-transfected control cells (Fig. 2a). In intracellular architecture is distinct. By superimposing a TRPC4 patch clamp experiments, englerin A potentiated whole-cell monomer with representative TRP monomers from each sub- currents in a dose-dependent manner for 293T cells expressing family, we found that the overall fold of TRPC4 is closest to that full-length and truncated TRPC4. In addition, currents were of TRPM4 (Fig. 3a). TRPC4 has marked similarities to TRPM4 in inhibited by the TRPC4 blocker ML204 and the non-selective the TMDs despite their different tissue functions and lack of blocker 2-APB (Fig. 2b). The current–voltage (I–V) relationships sequence conservation (<20% identical residues) (Supplementary from both constructs were typical of TRPC4/5, with flattening of Fig. 5a, b). Distinctive features of TRPC4 include: (1) the 2 NATURE COMMUNICATIONS | (2018) 9:3102 | DOI: 10.1038/s41467-018-05247-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05247-9 ARTICLE a 90° Side view Top view b 100 Å 100 Å 120 Å 100 Å 90° c d Disulfide bond Disulfide bond helix Pore S1 S2 S3 S6 S4 Pore S5 helix S4 Pre-S1 S2-S3 S1 Pre-S1 helix linker TRP domain Elbow Pre-S1 S2 S3 S5 elbow S6 TRP domain S2-S3 linker H4 H5 H6 H7 Connecting helix H3 H2 Connecting helix N-terminal domain HLH H1 AR4 AR4 Coiled-coil domain Coiled-coil domain N-terminal AR3 AR3 domain ARD AR2 AR2 AR1 AR1 Fig. 1 Overall structure of mTRPC4. a Side and top views of the cryo-EM density map of mouse TRPC4 at 3.3 Å overall resolution. Each monomer is represented in different colors. b Ribbon diagrams of the mouse TRPC4 model with channel dimensions indicated. c Ribbon diagrams depicting structural details of a single subunit. d Linear diagram depicting the major structural domains of the TRPC4 monomer, color-coded to match the ribbon diagram in (c) arrangement of S2–S3 linker, S5, S6, and the pore loop. In characteristic bridge loop (approximately 60 residues) connects TRPC4, the S2–S3 linker has two-helical turns, shorter than that the pre-S1 helix with the pre-S1 elbow (Fig. 3d and Supplemen- of TRPM4 (Supplementary Fig. 5a), which limits the interactions tary Fig. 5c). of S2 and S3 with their cytoplasmic regions; (2) the disulfide bond To understand the role of the disulfide bond in the pore loop of between TRPC4’s Cys549 and Cys554 lies in the loop linking S5 TRPC4, we performed patch clamp experiments on wild-type and the pore helix (Fig.