Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.
At a glance
- An introduction to TRP channels. Annu. Rev. Physiol. 68, 619–647 (2006) , &
- TRP channels. Annu. Rev. Biochem. 76, 387–417 (2007) &
- Transient receptor potential channelopathies. Pflugers Arch. 460, 437–450 (2010) &
- Gating of TRP channels: a voltage connection? J. Physiol. (Lond.) 567, 35–44 (2005) et al.
- Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005) , &
- Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007) , , &
- The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358 (2011) , , &
- Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012) et al.
- The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997) et al.
- TRPV1 channels are intrinsically heat sensitive and negatively regulated by phosphoinositide lipids. Neuron 77, 667–679 (2013) , , , &
- Kinetic and energetic analysis of thermally activated TRPV1 channels. Biophys. J. 99, 1743–1753 (2010) , &
- Targeting TRP channels for pain relief. Eur. J. Pharmacol. 716, 61–76 (2013) , &
- TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29, 355–384 (2013)
- Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013) , , &
- Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nature Methods 10, 584–590 (2013) et al.
- 3.88 Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy. Nature 453, 415–419 (2008) , &
- 3.3 Å cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry. Cell 141, 472–482 (2010) , , , &
- The TRPC3 channel has a large internal chamber surrounded by signal sensing antennas. J. Mol. Biol. 367, 373–383 (2007) et al.
- Structure of TRPV1 channel revealed by electron cryomicroscopy. Proc. Natl Acad. Sci. USA 105, 7451–7455 (2008) , , , &
- A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryoelectron microscopy. J. Biol. Chem. 285, 11210–11218 (2010) , , , &
- TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nature Neurosci. 11, 555–564 (2008) , &
- A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating. Neuron 58, 362–373 (2008) , &
- Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012) &
- The ankyrin repeats of TRPV1 bind multiple ligands and modulate channel sensitivity. Neuron 54, 905–918 (2007) , , , &
- Ion channel voltage sensors: structure, function, and pathophysiology. Neuron 67, 915–928 (2010)
- Sensing voltage across lipid membranes. Nature 456, 891–897 (2008)
- Conserved residues within the putative S4–S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels. J. Biol. Chem. 285, 41455–41462 (2010) , , &
- Protons stabilize the closed conformation of gain-of-function mutants of the TRPV1 channel. Biochim. Biophys. Acta 1833, 520–528 (2013) , &
- TRPV1 structures in distinct conformations reveal mechanisms of activation. Nature http://dx.doi.org/10.1038/nature12823 (this issue) , , &
- Structure–functional intimacies of transient receptor potential channels. Q. Rev. Biophys. 42, 201–246 (2009) , &
- Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome. Am. J. Hum. Genet. 90, 558–564 (2012) et al.
- Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005) , &
- Increased basal activity is a key determinant in the severity of human skeletal dysplasia caused by TRPV4 mutations. PLoS ONE 6, e19533 (2011) , &
- Permeation and selectivity of TRP channels. Annu. Rev. Physiol. 68, 685–717 (2006) , , &
- Contribution of the putative inner-pore region to the gating of the transient receptor potential vanilloid subtype 1 channel (TRPV1). J. Neurosci. 27, 7578–7585 (2007) , , , &
- Outer pore architecture of a Ca2+-selective TRP channel. J. Biol. Chem. 279, 15223–15230 (2004) , , &
- Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers. Nature 449, 607–610 (2007) , &
- Structural determinants of gating in the TRPV1 channel. Nature Struct. Mol. Biol. 16, 704–710 (2009) et al.
- The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998) et al.
- A primer on ankyrin repeat function in TRP channels and beyond. Mol. Biosyst. 4, 372–379 (2008)
- Structural diversity in the cytoplasmic region of G protein-gated inward rectifier K+ channels. Channels (Austin) 1, 39–45 (2007) , , &
- Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 Å resolution. Cell 111, 957–965 (2002) &
- Structural and biochemical consequences of disease-causing mutations in the ankyrin repeat domain of the human TRPV4 channel. Biochemistry 51, 6195–6206 (2012) , , &
- Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition. J. Vis. Exp. 58, 3227 (2011) , &
- BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies. Protein Expr. Purif. 62, 160–170 (2008) , , , &
- Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl Acad. Sci. USA 99, 13419–13424 (2002) , , &
- Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. EMBO J. 30, 4652–4664 (2011) , , &
- Amphipols from A to Z. Annu. Rev. Biophys. 40, 379–408 (2011) et al.
- Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003) &
- SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996) et al.
- FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007)
- RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)
- UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004) et al.
- Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004) &
- Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010) , , &
- HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996) , , , &
Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: A minimal TRPV1 channel that is functional and biochemically stable. (181 KB)
a, Mammalian (HEK293) cells expressing a minimal construct (with an N-terminal green fluorescent protein (GFP) tag) responded to various TRPV1 agonists, including capsaicin (Cap; 0.5 μM), extracellular protons (pH 5.0) and double-knot spider toxin (DkTx; 2 μM). Electrophysiological responses were measured in whole-cell patch-clamp configuration. b, c, Dose–responsive curves for capsaicin (b) or protons (c) were determined for minimal (black) or full-length (red) TRPV1, both of which contained an N-terminal GFP fusion. Values were normalized to maximal currents evoked by 30 μM capsaicin (b) or pH 4.0 (c) (n = 6 independent whole-cell recordings). d, DkTx dose–response curves for minimal (black) or full-length (red) TRPV1 as in b and c, determined by calcium imaging. Values were normalized to maximal capsaicin (10 μM)-evoked response in transfected HEK293 cells (n > 30 per point). e, Thermal response profiles for minimal (black) or full-length (red) TRPV1-expressing oocytes reveal similar heat sensitivity. f, Ion permeability ratios of agonist-evoked currents from minimal TRPV1 were estimated from reversal potential shifts in whole-cell patch-clamp recordings of transfected HEK293 cells, revealing no significant differences from full-length channel. g, Gel-filtration profile (Superdex-200) of detergent solubilized TRPV1 after purification on amylose affinity resin and proteolytic removal of maltose-binding protein (MBP) tag. The major species elutes as a symmetrical peak after the void volume (V0). Inset shows that peak material migrates as a single, homogeneous band on SDS–PAGE (4–12% gradient gel; Coomassie stain).
- Extended Data Figure 2: Sequence alignment of TRPV1 to other TRPV family members. (1,451 KB)
The rat TRPV1 construct used for this study consists of residues 110 to 764 (indicated by red arrows), excluding the highly divergent region (604–626, highlighted by cyan box). Secondary structure elements are indicated above the sequence. The starting points of six ankyrin repeats are based on a crystal structure of ARD of TRPV1 (PDB 2PNN). Several critical residues discussed in the text are labelled in blue, and conserved glycine and proline residues at the turn of a β-sheet (highlighted in Fig. 6) are indicated with red stars.
- Extended Data Figure 3: Negative-stain EM of TRPV1. (569 KB)
a, Representative negative-stain image of purified minimal TRPV1 protein in detergent (n-dodecyl β-d-maltopyranoside; DDM) after proteolytic removal of MBP tag. b, 2D class averages of negatively stained particles in DDM. c, d, Two views of a random conical tilt (RCT) reconstruction from negatively stained TRPV1 in DDM. The RCT reconstruction was low-pass filtered at 30 Å, and fitted with the structure of NaVAb (PDB 3RVY) to indicate the size and general shape. e, Gel-filtration profile (Superdex-200) of purified minimal TRPV1 protein after exchange from DDM into amphipols. The major species elutes as a symmetrical peak after the void volume (V0). f, Representative negative-stain image of purified minimal TRPV1 protein without MBP tag in amphipols. g, 2D class averages of negative-stain particles in amphipols.
- Extended Data Figure 4: Cryo-EM of TRPV1 using Tecnai TF20 microscope and TemF816 8k × 8k CMOS camera. (1,000 KB)
a–d, Representative images of frozen hydrated TRVP1 in amphipols taken at different defocus levels, 3.1 μm (a) and 1.5 μm (b) and their Fourier transforms (c, d). Thon rings extend to ~8 Å. Dash-line squares or circles indicate representative particles showing two distinctive views. e, 2D class averages of TRPV1 particles. f, Enlarged view of three representative 2D class averages.
- Extended Data Figure 5: 3D reconstruction of TRPV1 calculated from TF20 data. (370 KB)
a, Gold-standard FSC curve for the 3D reconstruction, marked with resolutions corresponding to FSC = 0.5 and 0.143. b, Side view of the 3D reconstruction low-pass filtered at 9 Å and amplified by a temperature factor −1,500 Å2, showing transmembrane (top) and cytoplasmic (bottom) domains. The transmembrane domain roughly fitted by the atomic model of NaVAb (PDB 3RVY). c, Longitudinal cross section view focused on central transmembrane helices. d, Bottom-up view of the 3D reconstruction shows overall structure. e, f, Bottom-up cross-section views showing the arrangement of transmembrane (e) and cytoplasmic (f) domains.
- Extended Data Figure 6: Motion correction improves the quality of images collected on Polora TF30 microscope using a K2 Summit direct electron detector. (693 KB)
a, Fourier transform of a representative cryo-EM image of TRPV1 embedded in a thin layer of vitreous ice over Quantifoil hole without supporting carbon film before motion correction. b, Path of motion of 30 individual subframes, determined as described in Methods. c, d, A nearly perfect Fourier transform (c) was restored after the EM image was corrected for motion (d).
- Extended Data Figure 7: Picking and 2D classification of TRPV1 Cryo-EM particles collected on Polora TF30 microscope. (783 KB)
a, Representative cryo-EM image after motion correction. Green boxes indicate all particles that were selected by semi-automatic particle picking and 2D screening, as described in Methods. b, Gallery view of the particles shown in a. c, 2D class averages of cryo-EM particles show many fine features (also seen in enlarged views in Fig. 1c), and these features are not visible in the 2D class averages of cryo-EM particles from TF20 data (Extended Data Fig. 4e, f).
- Extended Data Figure 8: 3D reconstruction of TRPV1 calculated from TF30 data. (521 KB)
a, Gold-standard FSC curve (red) of the final 3D reconstruction, marked with resolutions corresponding to FSC = 0.5 and 0.143. The FSC curve between the final map and that calculated from the atomic model is shown in blue. The relative low value of this FSC (blue) at low frequency range (>10 Å) is probably due to the presence of amphipol density in the experimental map. b, Euler angle distribution of all particles used for calculating the final 3D reconstruction. The sizes of balls represent the number of particles. The accuracy of rotation is 3.54°, as reported by RELION. c, Different views of the 3D reconstruction low-pass filtered at 6 Å and amplified by a temperature factor of −100 Å2, fitted with the atomic model of TRPV1. d, Two views of the 3D reconstruction displayed at two different isosurface levels (high in yellow and low in grey). At the low isosurface level, the belt-shaped density of amphipols is visible with a thickness of ~30 Å.
- Extended Data Figure 9: Cryo-EM densities of selected regions of TRPV1 at 3.4 Å resolution. (579 KB)
a–d, Representative cryo-EM densities (grey mesh) are superimposed on atomic model (main chain in pink) for various TRPV1 domains, as indicated. e, f, Representative cryo-EM densities (grey mesh) are docked with crystal structure of TRPV1 ankyrin repeats (PDB 2PNN). Accuracy of docking was supported by fitting of several bulky side chains. Map was low-pass filtered to 3.4 Å and amplified by a temperature factor −100 Å2.
- Extended Data Figure 10: Details of domain interactions and outer pore configurations. (606 KB)
a–d, Cryo-EM densities (grey mesh) of highlighted regions of TRPV1, as indicated, at 3.4 Å resolution are superimposed onto atomic model. Map was low-pass filtered to 3.4 Å and amplified by a temperature factor −100 Å2. e, Superimposition of TRPV1 (salmon) with KV 1.2–2.1 chimaera (PDB 2R9R; grey). f, Superimposition of TRPV1 (salmon) with NaVAb (PDB 3RVY; blue). In each case, substantial structural differences are observed in the outer pore region. Structural alignments are based on the pore domain (S5–P–S6).